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YTISREVINUALAGAPPA
APPAGALAUNIVERSITY
M.Sc. [Microbiology] elcyC drihT eht ni )46.3:APGC( CA[Accredited
AN yb edarGwith
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364 22
]CGU-DRHM yb ytisrevinU I–yrogeand
taC Graded
sa dedarasG Category–I
dna University by MHRD-UGC]
300 036 – IDUKIARA
KARAIKUDI
K – 630 003
NOITACUDE ECNATSIDDIRECTORATE
FO ETAROTCEOF
RIDDISTANCE EDUCATION
MOLECULAR BIOLOGY AND
rDNA TECHNOLOGY

MOLECULAR BIOLOGY AND rDNA TECHNOLOGY

II - Semester

M.Sc. [Microbiology]
364 22

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TISREVINUALAGAPPA
APPAGALAUNIVERSITY
M.Sc. [Microbiology]
lcyC drihT eht ni )46.3:APGC( CA[Accredited
AN yb edarGwith
’+A’’A+’
htiwGrade
detidby
ercNAAC
cA[ (CGPA:3.64) in the Third Cycle
]CGU-DRHM yb ytisrevinU I–yrogeand
300 036 – IDUKIARA
KARAIKUDI
K
taC Graded
sa dedaras
– 630 003
G Category–I
dna University by MHRD-UGC]
MOLECULAR BIOLOGY AND
NOITACUDE ECNATSIDDIRECTORATE
FO ETAROTCEOF
RIDDISTANCE EDUCATION
rDNA TECHNOLOGY
II - Semester
 ALAGAPPA UNIVERSITY
[Accredited with ‘A+’ Grade by NAAC (CGPA:3.64) in the Third Cycle
and Graded as Category–I University by MHRD-UGC]
(A State University Established by the Government of Tamil Nadu)
KARAIKUDI – 630 003

Directorate of Distance Education

M.Sc. (Microbiology)
II - Semester
364 22

MOLECULAR BIOLOGY AND

rDNA TECHNOLOGY
Authors
Dr. Neeti Pandey, Assistant Professor, Department of Zoology, SGTB Khalsa College, University of Delhi
Units (1, 2.3, 4, 5, 6)
Dr. Pradeep Kumar, Research Associate-III, Department of Pediatrics, Army Hospital Research & Referral, New Delhi
Units (2.2, 2.4, 2.5, 3.2)
Dr. Soumya Mukherjee, Assistant Professor, Department of Botany, Jangipur College, University of Kalyani, West Bengal
Units (7-14)
Vikas Publishing House, Units (2.0-2.1, 2.5.1-2.10, 3.0-3.1, 3.3-3.7)
"The copyright shall be vested with Alagappa University"

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 SYLLABI-BOOK MAPPING TABLE
MOLECULAR BIOLOGY AND rDNA TECHNOLOGY
Syllabi Mapping in Book

BLOCK-1: DNA AND ITS REPLICATION, RNA AND ITS TYPES

Unit I
Discovery of DNA- Molecular Basis of DNA as Genetic Material. Structure Unit 1: DNA: Discovery,
of DNA - A, B and Z Form. Forms of DNA - DNA Heteroduplex, Circular, Structure, Forms and Properties
Superhelical DNA, Twisted Circle. Properties of DNA - Denaturation, (Pages 1-40)
Renaturation, Melting Curve and Hyperchromicity. Unit 2: DNA: Replication,
Unit II Enzymology and Types
Replication of DNA- Semi Conservative Mode, Meselson - Stahl Experiment. (Pages 41-80)
Enzymology of DNA Replication - DNA Polymerase I, II & III, Topoisomerase Unit 3: RNA: Structure and
I & II, Helicase, Primase, Gyrase. Molecular Basis of DNA Replication - Types
Replication Fork, Origin, Okazaki Fragments. Types of Replication - Circular (Pages 81-94)
and Theta.
Unit III
Structure of RNA - Types of RNA - tRNA, mRNA and rRNA.

BLOCK-2: PROTEIN SYNTHESIS & CLONING VECTORS

Unit IV
Transcription Process in Prokaryotes- Initiation - Promotors, Upstream and Unit 4: Process of Transcription
Downstream Sequences, Transcription Factors. Elongation - RNA Polymerase, in Prokaryotes
Sub Units. Termination - Rho Dependent and Rho Independent, NusA Protein. (Pages 95-106)
Antitermination. Unit 5: RNA Processing
Unit V (Pages 107-118)
RNA Processing (Post Transcriptional Modifications), Inhibitors of Unit 6: Cloning Vectors
Transcription. Reverse Transcription. (Pages 119-146)
Unit VI
Cloning Vectors - Plasmids, Cosmids, Phasmids, Phagemids, Expression
Vectors, Plasmid Vectors - pBR322 and pUC18, Integrating Shuttle Vector -
YAC Vectors, Viral Vector - SV 40 and Adenovirus.

BLOCK-3: GENE CLONING

Unit VII
Cloning of Human Insulin, Interferon in E. coli. Recombinant Vaccine Unit 7: Cloning of Human
Development - HBs Ag in Yeast. Insulin (Pages 147-162)
Unit VIII Unit 8: Cloning for Commercial
Cloning for Commercial Production of Antibiotics (Penicillin). Production of Antibiotics
Unit IX (Pages 163-174)
Cloning Methodologies- D Complementation, Sticky and Blunt End Cloning. Unit 9: Cloning Methodologies
Cloning from mRNA - Synthesis of cDNA, Cloning cDNA- cDNA Library. (Pages 175-198)
Cloning from Genomic DNA - Genomic Library. Shot Gun Cloning.
Unit X Unit 10: Screening of
Screening of Recombinant - Phenotypic Expression of Characters. Blotting Recombinants, Blotting
Techniques - Western, Northern and Southern. Techniques and Blotting
Mapping of Human Genes - Human Genome Project. Techniques (Pages 199-224)

BLOCK-4: MOLECULAR TECHNIQUES

Unit XI Unit 11: PCR Methods and Primer
PCR- Gene Amplification, Primer Designing, Optimization, Variation in the Designing
PCR (RAPD and RFLP) (Pages 225-252)
Unit XII Unit 12: DNA Sequencing
DNA Sequencing - Sanger - Coulson's Method, Maxam-Gilbert's Method, Methods
Automated Sequencing and Micro Array. (Pages 253-272)
Unit XIII Unit 13: Gene Silencing and
Gene Silencing and Antisense Technology- Types and Mechanism of Gene Antisense Technology
Silencing. Genetic Factors of Silencing, Formation of Antisense mRNA, (Pages 273-294)
Inhibition of Gene Expression by Antisense RNA. Unit 14: Plant Genetic
Unit XIV Engineering
Plant Genetic Engineering- Ti Plasmid, CaMV Vector, Direct DNA Delivery (Pages 295-318)
Methods - Micro Projectile Bombardment, Microinjection and Electroporation.
 CONTENTS
INTRODUCTION

BLOCK I: DNA AND ITS REPLICATION, RNA AND ITS TYPES

UNIT 1 DNA: DISCOVERY, STRUCTURE, FORMS AND PROPERTIES 1-40
1.0 Introduction
1.1 Objectives
1.2 DNA: Introduction and Discovery
1.2.1 Molecular Basis of DNA
1.2.2 Properties and Characteristics of DNA
1.2.3 Conformations of DNA Double Helix
1.2.4 Forms of DNA
1.3 Answers to Check Your Progress Questions
1.4 Summary
1.5 Key Words
1.6 Self Assessment Questions and Exercises
1.7 Further Readings

UNIT 2 DNA: REPLICATION, ENZYMOLOGY AND TYPES 41-80

2.0 Introduction
2.1 Objectives
2.2 General Introduction
2.3 DNA Replication: An Insight
2.4 Enzymology of DNA Replication
2.5 DNA Replication Mechanism: Prokaryotic and Eukaryotic
2.5.1 Types of Replication
2.6 Answers to Check Your Progress Questions
2.7 Summary
2.8 Key Words
2.9 Self Assessment Questions and Exercises
2.10 Further Readings

UNIT 3 RNA: STRUCTURE AND TYPES 81-94

3.0 Introduction
3.1 Objectives
3.2 RNA: Structure, Function and Types
3.3 Answers to Check Your Progress Questions
3.4 Summary
3.5 Key Words
3.6 Self Assessment Questions and Exercises
3.7 Further Readings
BLOCK II: PROTEIN SYNTHESIS & CLONING VECTORS
UNIT 4 PROCESS OF TRANSCRIPTION IN PROKARYOTES 95-106
4.0 Introduction
4.1 Objectives
 4.2 Process of Transcription in Prokaryotes
4.2.1 Process of Transcription
4.2.2 Mechanism of Transcription
4.2.3 Prokaryotic vs. Eukaryotic Transcription
4.3 Answers to Check Your Progress Questions
4.4 Summary
4.5 Key Words
4.6 Self Assessment Questions and Exercises
4.7 Further Readings
UNIT 5 RNA PROCESSING 107-118
5.0 Introduction
5.1 Objectives
5.2 RNA Processing
5.3 Answers to Check Your Progress Questions
5.4 Summary
5.5 Key Words
5.6 Self Assessment Questions and Exercises
5.7 Further Readings

UNIT 6 CLONING VECTORS 119-146

6.0 Introduction
6.1 Objectives
6.2 Cloning Vectors
6.2.1 Plasmids as Cloning Vectors
6.2.2 Phage O
6.2.3 Cosmid Vectors
6.2.4 Phagemids
6.2.5 Vectors Required for the Cloning of Larger DNA Segments
6.2.6 Yeast Artificial Chromosomes (YAC Vectors)
6.2.7 Vectors for the Preparation of ssDNA
6.2.8 Viral Vector – SV 40 and Adenovirus
6.3 Answers to Check Your Progress Questions
6.4 Summary
6.5 Key Words
6.6 Self Assessment Questions and Exercises
6.7 Further Readings
BLOCK III: GENE CLONING
UNIT 7 CLONING OF HUMAN INSULIN 147-162
7.0 Introduction
7.1 Objectives
7.2 Cloning of Human Insulin
7.2.1 Methodology
7.2.2 Synthesis of Interferon in Escherichia coli
7.2.3 Development of Recombinant Vaccines
7.3 Answers to Check Your Progress Questions
7.4 Summary
7.5 Key Words
7.6 Self Assessment Questions and Exercises
7.7 Further Readings
UNIT 8 CLONING FOR COMMERCIAL PRODUCTION OF ANTIBIOTICS 163-174
8.0 Introduction
8.1 Objectives
8.2 Cloning for Commercial Production of Antibiotics
8.3 Answers to Check Your Progress Questions
8.4 Summary
8.5 Key Words
8.6 Self Assessment Questions and Exercises
8.7 Further Readings
UNIT 9 CLONING METHODOLOGIES 175-198
9.0 Introduction
9.1 Objectives
9.2 Cloning Methodologies
9.2.1 D (Alpha) Complementation Test
9.2.2 Sticky and Blunt End Cloning
9.2.3 Blunt End Cloning Method
9.2.4 Cloning From mRNA
9.2.5 Cloning from Genomic DNA Library
9.2.6 Cloning of Genomic DNA
9.2.7 Shotgun Cloning
9.3 Answers to Check Your Progress Questions
9.4 Summary
9.5 Key Words
9.6 Self Assessment Questions and Exercises
9.7 Further Readings

UNIT 10 SCREENING OF RECOMBINANTS, BLOTTING TECHNIQUES

AND BLOTTING TECHNIQUES 199-224
10.0 Introduction
10.1 Objectives
10.2 Screening of Recombinants: Phenotypic Expression of Characters
10.3 Blotting Techniques
10.4 Mapping of Human Genes: Human Genome Project
10.5 Answers to Check Your Progress Questions
10.6 Summary
10.7 Key Words
10.8 Self Assessment Questions and Exercises
10.9 Further Readings

BLOCK IV: PCR METHODS AND PRIMER DESIGNING

UNIT 11 225-252
11.0 Introduction
11.1 Objectives
11.2 Polymerase Chain Reaction (PCR)
11.2.1 Components of PCR Reaction Procedure
11.2.2 Steps of Polymerase Chain Reaction
11.3 Primer Designing and Optimization
11.3.1 Procedure for Primer Designing
 11.4 Variations in PCR
11.4.1 Random Amplification of Polymorphic DNA (RAPD): PCR Variant
11.4.2 Restriction Fragment Length Polymorphisms (RFLP)
11.5 Answers to Check Your Progress Questions
11.6 Summary
11.7 Key Words
11.8 Self Assessment Questions and Exercises
11.9 Further Readings
UNIT 12 DNA SEQUENCING METHODS 253-272
12.0 Introduction
12.1 Objectives
12.2 DNA Sequencing: An Introduction
12.2.1 Sanger Sequencing
12.2.2 Sanger and Coulson Dideoxynucleotide Synthetic Method
12.2.3 Maxam–Gilbert’s Method of Sequencing
12.2.4 Automated Sequencing Method
12.2.5 DNA Microarray Analysis
12.3 Answers to Check Your Progress Questions
12.4 Summary
12.5 Key Words
12.6 Self Assessment Questions and Exercises
12.7 Further Readings
UNIT 13 GENE SILENCING AND ANTISENSE TECHNOLOGY 273-294
13.0 Introduction
13.1 Objectives
13.2 Gene Silencing: An Introduction
13.2.1 Transcriptional Gene Silencing (TGS)
13.2.2 Genetic Factors Associated with Transcriptional Gene Silencing (TGS)
13.2.3 Post-Transcriptional Gene Silencing (PTGS)
13.3 Formation of Antisense mRNA and Inhibition of Gene Expression
13.4 Answers to Check Your Progress Questions
13.5 Summary
13.6 Key Words
13.7 Self Assessment Questions and Exercises
13.8 Further Readings
UNIT 14 PLANT GENETIC ENGINEERING 295-318
14.0 Introduction
14.1 Objectives
14.2 Basics of Plant Genetic Engineering
14.2.1 Ti Plasmid and Agrobacterium Mediated Gene Transfer
14.2.2 CaMV Vector and Its Applications
14.2.3 Direct DNA Delivery Methods
14.2.4 Micro Projectile Bombardment
14.2.5 Microinjection
14.2.6 Electroporation
14.3 Answers to Check Your Progress Questions
14.4 Summary
14.5 Key Words
14.6 Self Assessment Questions and Exercises
14.7 Further Readings
 Introduction
INTRODUCTION

Molecular biology is a branch of biology that concerns the molecular basis of

biological activity between biomolecules in the various systems of a cell, including NOTES
the interactions between DNA, RNA, proteins and their biosynthesis, as well as
the regulation of these interactions. Fundamentally, molecular biology is the study
of biology at a molecular level. The field overlaps with other areas of biology and
chemistry, particularly genetics and biochemistry. Biochemistry is the study of the
chemical substances and vital processes occurring in live organisms. Biochemists
focuses on the role, function, and structure of biomolecules. The study of the
chemistry behind biological processes and the synthesis of biologically active
molecules are examples of biochemistry. Genetics is the study of the effect of
genetic differences in organisms. This can often be inferred by the absence of a
normal component, such as one gene. The study of 'mutants' - the organisms
which lack one or more functional components with respect to the so-called 'wild
type' or 'normal phenotype'. Molecular biology refers to the study of molecular
underpinnings of the processes of replication, transcription, translation, and cell
function.
Characteristically, the molecular biology is concerned with the interactions
between the various systems of a cell, including the interrelationship of DNA,
RNA and protein synthesis and learning how these interactions are regulated. One
of the most basic techniques of molecular biology is to study protein function
specifically the molecular cloning. In this technique, DNA coding for a protein of
interest is cloned using Polymerase Chain Reaction (PCR), and/or restriction
enzymes into a plasmid (expression vector). A vector has 3 distinctive features -
an origin of replication, a Multiple Cloning Site (MCS), and a selective marker
usually antibiotic resistance. Introducing DNA into Eukaryotic cells, such as animal
cells, by physical or chemical means is called transfection. Several different
transfection techniques are available, such as calcium phosphate transfection,
electroporation, microinjection and liposome transfection. Large quantities of a
protein can then be extracted from the Bacterial or Eukaryotic Cell. The protein
can be tested for enzymatic activity under a variety of situations, the protein may
be crystallized so its tertiary structure can be studied and in the pharmaceutical
industry the activity of new drugs against the protein can be studied.
Polymerase Chain Reaction (PCR) is an extremely versatile technique for
copying DNA. In brief, PCR allows a specific DNA sequence to be copied or
modified in predetermined ways. The reaction is extremely powerful and under
perfect conditions could amplify one DNA molecule to become 1.07 billion
molecules in less than two hours. The PCR technique can be used to introduce
restriction enzyme sites to ends of DNA molecules and to determine whether a
particular DNA fragment is found in a cDNA library. PCR has many variations,
like Reverse Transcription PCR (RT-PCR) for amplification of RNA, and, more
Self-Instructional
Material 9
Introduction recently, quantitative PCR which allow for quantitative measurement of DNA or
RNA molecules.
This book, Molecular Biology & rDNA Technology, is divided into four
blocks that are further divided into fourteen units which will help to understand the
NOTES
concepts of molecular biology and rDNA Technology, such as DNA and its
replication, RNA and its types, discovery of DNA, molecular basis of DNA as
genetic material, structure of DNA, forms of DNA, properties of DNA, replication
of DNA, Meselson-Stahl experiment, enzymology of DNA replication, DNA
polymerase (I, II and III), molecular basis of DNA replication, Okazaki fragments,
types of replication - circular and theta, structure of RNA, types of RNA (tRNA,
mRNA and rRNA), protein synthesis and cloning vectors, transcription process
in Prokaryotes, transcription factors, elongation, RNA polymerase, sub units, RNA
processing (post transcriptional modifications), inhibitors of transcription, reverse
transcription, cloning vectors (plasmids, cosmids, phasmids, phagemids), plasmid
vectors - pBR322 and pUC18, YAC vectors, viral vector - SV 40 and adenovirus,
gene cloning, cloning of human insulin, recombinant vaccine development - HBs
Ag in yeast, cloning for commercial production of antibiotics (Penicillin), cloning
methodologies, cloning from mRNA, synthesis of cDNA, cloning cDNA, cloning
from genomic DNA, genomic library, shotgun cloning, screening of recombinant,
phenotypic expression of characters, blotting techniques (western, northern and
southern), mapping of human genes, molecular techniques, PCR, gene amplification,
primer designing, optimization, variation in the PCR (RAPD and RFLP), DNA
sequencing, Sanger-Coulson method, Maxam-Gilbert method, automated
sequencing and microarray, gene silencing and antisense technology, types and
mechanism of gene silencing, genetic factors of silencing, formation of antisense
mRNA, plant genetic engineering- Ti plasmid, CaMV vector, direct DNA delivery
methods, micro projectile bombardment, microinjection and electroporation.
The book follows the self-instruction mode or the SIM format wherein
each unit begins with an 'Introduction' to the topic followed by an outline of the
'Objectives'. The content is presented in a simple, organized and comprehensive
form interspersed with 'Check Your Progress' questions and answers for better
understanding of the topics covered. A list of 'Key Words' along with a 'Summary'
and a set of 'Self Assessment Questions and Exercises' is provided at the end of
the each unit for effective recapitulation.

Self-Instructional
10 Material
 DNA: Discovery,
BLOCK - I Structure, Forms and
Properties
DNA AND ITS REPLICATION, RNA AND ITS TYPES

NOTES
UNIT 1 DNA: DISCOVERY,
STRUCTURE, FORMS AND
PROPERTIES
Structure
1.0 Introduction
1.1 Objectives
1.2 DNA: Introduction and Discovery
1.2.1 Molecular Basis of DNA
1.2.2 Properties and Characteristics of DNA
1.2.3 Conformations of DNA Double Helix
1.2.4 Forms of DNA
1.3 Answers to Check Your Progress Questions
1.4 Summary
1.5 Key Words
1.6 Self Assessment Questions and Exercises
1.7 Further Readings

1.0 INTRODUCTION

DeoxyriboNucleic Acid (DNA) is a molecule composed of two chains that coil

around each other to form a double helix carrying genetic instructions for the
development, functioning, growth and reproduction of all known organisms and
many viruses. DNA and RiboNucleic Acid (RNA) are nucleic acids; alongside
proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are
one of the four major types of macromolecules that are essential for all known
forms of life. The two DNA strands are also known as polynucleotides as they are
composed of simpler monomeric units called nucleotides. Each nucleotide is
composed of one of four nitrogen-containing nucleobases (Cytosine [C], Guanine
[G], Adenine [A] or Thymine [T]), a sugar called deoxyribose, and a phosphate
group. The nucleotides are joined to one another in a chain by covalent
bonds between the sugar of one nucleotide and the phosphate of the next, resulting
in an alternating sugar-phosphate backbone. The nitrogenous bases of the two
separate polynucleotide strands are bound together, according to base pairing rules
(A with T and C with G), with hydrogen bonds to make double-stranded DNA.
The complementary nitrogenous bases are divided into two groups, pyrimidines
and purines. In DNA, the pyrimidines are thymine and cytosine; the purines are
adenine and guanine.
Self-Instructional
Material 1
DNA: Discovery, Both strands of double-stranded DNA store the same biological information.
Structure, Forms and
Properties This information is replicated as and when the two strands separate. A large part
of DNA (more than 98% for humans) is non-coding, meaning that these sections
do not serve as patterns for protein sequences. The two strands of DNA run in
NOTES opposite directions to each other and are thus antiparallel. Attached to each sugar
is one of four types of nucleobases. It is the sequence of these four nucleobases
along the backbone that encodes genetic information. RNA strands are created
using DNA strands as a template in a process called transcription. Under the genetic
code, these RNA strands specify the sequence of amino acids within proteins in a
process called translation.
Within eukaryotic cells, DNA is organized into long structures called
chromosomes. Before typical cell division, these chromosomes are duplicated in
the process of DNA replication, providing a complete set of chromosomes for
each daughter cell. Eukaryotic organisms (animals, plants, fungi and protists) store
most of their DNA inside the cell nucleus as nuclear DNA, and some in
the mitochondria as mitochondrial DNA or in chloroplasts as chloroplast DNA. In
contrast, Prokaryotes (Bacteria and Archaea) store their DNA only in
the cytoplasm, in circular chromosomes. Within eukaryotic chromosomes,
chromatin proteins, such as histones, compact and organize DNA. These
compacting structures guide the interactions between DNA and other proteins,
helping control which parts of the DNA are transcribed.
DNA was first isolated by Friedrich Miescher in 1869. Its molecular
structure was first identified by Francis Crick and James Watson at the Cavendish
Laboratory within the University of Cambridge in 1953, whose model-building
efforts were guided by X-ray diffraction data acquired by Raymond Gosling, who
was a post-graduate student of Rosalind Franklin. DNA is used by researchers as
a molecular tool to explore physical laws and theories, such as the ergodic
theorem and the theory of elasticity. The unique material properties of DNA have
made it an attractive molecule for material scientists and engineers interested in
micro- and nanofabrication. Among notable advances in this field are DNA
origami and DNA-based hybrid materials.
In this unit, you will study about DNA, its discovery, structure, forms and
properties in detail.

1.1 OBJECTIVES

After going through the unit, you will be able to:

• Understand the structure of DNA
• Explain DNA as genetic material
• Discuss the various forms of DNA
• Analyse the functions of DNA
Self-Instructional
2 Material
 DNA: Discovery,
1.2 DNA: INTRODUCTION AND DISCOVERY Structure, Forms and
Properties

DNA is the chemical name for the molecule that carries genetic instructions in all
living things. The DNA molecule consists of two strands that wind around one NOTES
another to form a shape known as a double helix. Each strand has a backbone
made of alternating sugar (deoxyribose) and phosphate groups. Attached to each
sugar is one of four bases: Adenine (A), Cytosine (C), Guanine (G), and Thymine
(T). The two strands are held together by bonds between the bases; Adenine
bonds with Thymine, and Cytosine bonds with Guanine. The sequence of the
bases along the backbones serves as instructions for assembling protein and RNA
molecules.
Nucleic acids are of two types- DeoxyriboNucleic Acid (DNA) and
RiboNucleic Acid (RNA), both of which primarily serve as reservoir and transmitters
of genetic information. Johann Friedrich Miescher, a Swiss researcher, discovered
DNA in 1869. Avery, Macleod and McCarty first demonstrated in 1944 that
DNA contained genetic information. Friedrich Miescher (1869) first isolated nucleic
acids from pus cells. Initially named nuclein, Hertwig (1884) believed them to be
the carrier of hereditary traits. Owing to their acidic nature the term nucleinic acids
was used for them which was later replaced by nucleic acids (Altmann, 1899).
The presence of purine and pyrimidine bases in nucleic acids was discovered by
Fisher (1880s). Deoxyribose nucleic acid was found to contain phosphoric acid
as well as deoxyribose sugar by Levene (1910) who also gave the characterization
of 4 types of nucleotides present in DNA. In 1950, Chargaff found the content of
purine and pyrimidine present in DNA to be equal. By this time W.T. Astbury had
discovered through X-ray diffraction that DNA is a polynucleotide in which
nucleotides are arranged perpendicular to the long axis of the molecule and spaced
at a distance of 0.34 nm. Significant information about the structure of DNA was
brought to light in 1953 by the very fine X-ray photographs of DNA taken by
Wilkins and Franklin. The photographs showed that DNA was present in the form
of a helix of width 2.0 nm. One turn of the helical structure was 3.4 nm comprising
of 10 layers of bases which were stacked in it. The double helix model was first
correctly worked out by Watson and Crick (1953) from the X-ray photographs
of Wilkins and Franklin. Nobel Prize was awarded to Wilkins, Watson and Crick
for the same in 1962. In 1953 Watson and Crick developed a 3D, molecular
model of DNA that satisfied all the minute details obtained from X-ray photographs.
Therefore they proposed that DNA comprised of a double helix which had two
chains consisting of sugar phosphate on the outside and nitrogen bases on the
inner core/side. A hall mark of their proposition complementary base pairing between
the two polynucleotide chains where the complementary pairs formed between
purine of one and pyrimidine of the other chain were held together via hydrogen
bonds (A-T, C-G). Their second important proposal was that the two chains are
antiparallel, i.e., while one had 5´ → 3´ orientation of one, the other had Y → 5´
orientation. This unique double helical or duplex model of DNA having antiparallel
Self-Instructional
Material 3
DNA: Discovery, polynucleotide chains with complementary bases has a fundamental mechanism of
Structure, Forms and
Properties its replication and copying. Here both the polynucleotide chains work as templates
resulting in the formation of two double helices where each has one parent chain
and one new but complementary strand. The phenomenon is known as semi
NOTES conservative replication. Kornberg in 1959 carried out the in vitro synthesis of
DNA.
1.2.1 Molecular Basis of DNA
Chemical analysis of highly purified DNA has shown that it is made of four kinds
of monomeric building blocks, each of which contains three types of molecules:
• Phosphoric Acid
• Pentose Sugar
• Organic Bases
Phosphoric Acid

The phosphoric acid (H3PO4) is biologically called phosphate and it was discovered
by Levene in 1910. Phosphoric acid consists of three reactive hydroxyl groups
(–OH), out of which two are involved in forming sugar phosphate backbone of
both DNA as well as RNA. A phosphate group binds to the 5´ carbon of one and
3´ carbon of the other adjacent pentose sugar molecule to make the phosphate
diester. The phosphate makes a nucleotide negatively charged.
Therefore, a DNA becomes a polyanionic structure (Refer Figure 1.1):

Fig. 1.1 Structure of Phosphoric Acid

Pentose Sugar
DNA contains β D 2´ -deoxyribose sugar. It is a five-carbon sugar; hence it is a
pentose sugar. Since one oxygen atom at the 2´ carbon is missing it get its name
2´ -deoxy. The 2´ - deoxy-containing backbone is more resistant to hydrolysis as
compared to the riboform.
D-ribose does not mean dextrorotary ribose. It is actually a form of
stereoisomer. Here the prefix D is used to refer to the configuration of sugar due
to presence of asymmetric carbon atom. Any D-sugar is the mirror image of
L-sugar with respect to the orientation and the position of monovalent atom or
group linked with asymmetric carbon atom, i.e., monovalent atom or group in one
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form of sugar is exactly opposite in position (left- right) in other form of sugar. In DNA: Discovery,
Structure, Forms and
D-ribose there are three asymmetric carbon atoms. Properties
In deoxyribose sugar, the hydroxyl group on the carbon that carries the
aldehyde group can rapidly change from one position to another. The two positions
NOTES
are known as α and β.
The ring form in which the deoxyribose sugar is always present is derived
from heterocyclic furan (C4H40) structure. The carbon atoms of the deoxyribose
are numbered from the end closet to the aldehyde and the numbers are given as 1´,
2´, 3´, 4´ and 5´ in order to distinguish and characterise them from the corresponding
position in DNA bases. It is also explained that each numbered carbon on the
sugar is followed by a prime mark, therefore one speaks of 5 prime or 3 prime
carbon, etc. (Refer Figure 1.2).

Fig. 1.2 Structure of Sugars Present in Nucleic Acids

Organic Bases
Many varying kinds of heterocyclic nitrogen comprising of ring compounds are
found in the structure of DNA. They are known as simply bases because they can
combine with H+ in acidic solution. They are also referred to as nitrogenous base
because of the presence of nitrogen.
The organic bases of DNA can be characterised into two major groups:
• Pyrimidines.
• Purines—on the basis of their structures.
Pyrimidines
Pyrimidine bases are composed of a six-membered pyrimidine ring, which is similar
in structure to the benzene ring except that it contains nitrogen in place of carbon at
the positions of 1 and 3. Three pyrimidine derivatives are Uracil, Thymine and
Cytosine.
Their names are generally abbreviated by three capital letters, viz., U, T and
C, respectively. While Cytosine and Thymine are commonly found in DNA,
Cytosine and Uracil are found in RNA. In RNA, Thymine is replaced by Uracil.
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DNA: Discovery, Thymine is characteristically present in DNA because Thymine ensures
Structure, Forms and
Properties stability of the genetic message. Otherwise retention of the Uracil would result in
mispairing and mutagenesis on subsequent replication.
All pyrimidine bases consist of common keto-oxygen at position 2. In
NOTES
cytosine, an amino group (-NH2) is present at position 4. An additional keto-
oxygen is present at position 4 in Uracil. But Thymine has a keto-oxygen at position
4 and a methyl group (CH3) at position 5. All pyrimidine bases have a hydrogen
atom at the position 1, which is involved in their linkage with carbon 1 of pentose
sugar.
Purines
Purine is a derivative of pyrimidine which comprises of a pyrimidine ring and a
five-membered imidazole ring (having nitrogen at 7 and 9 positions) which are
fused together at 5 and 4 position.
There are two purine compounds namely Adenine (A) and Guanine (G) which are
found in the structure of DNA. Adenine has an amino group (-NH2) at 6 position
while guanine has a keto group and an amino group at, 6 and 2 positions of
carbon, respectively. The pentose sugar is linked to the base via a β-N glucosidic
bond between carbon atom 1 of the pentose and nitrogen atom 9 of purine bases
(Refer Figure 1.3).

Fig, 1.3 Structure of Purines and Pyrimidines Found in Nucleic Acids

Rare or Minor Bases

In addition to four common bases (ATGC), certain other unusual bases of purine
and pyrimidine derivatives, called rare or minor bases, occur in small amounts in
some DNA. In some viruses Uracil occurs in place of Thymine in DNA.
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 The T-even phages contain 5-hydroxy methyl cytosine in place of Cytosine. DNA: Discovery,
Structure, Forms and
Moreover, the hydroxyl group of hydroxyl-methyl Cytosine is often glucosylated Properties
(Refer Figure 1.4). These modifications protect the viral DNA from degradation
by the host cell endonucleases.
NOTES

Fig. 1.4 Structure of 5-Hydroxy Methyl Cytosine

General Properties of Bases

All pyrimidines and purines have some general properties:
• Free pyrimidine and purine bases are relatively insoluble in water.
• Free pyrimidine and purine occur only in trace amounts in most cells.
• They are weakly basic compounds that may exist in two or more tautomeric
forms depending upon the pH.
• They strongly absorb ultraviolet light in the region 250 to 260 nm. The
alternating double and single bonds between the carbon atoms in the
nitrogenous bases can interchange continuously, producing resonance. As a
result, the bases absorb ultraviolet light at 260 nm. This property is very
useful in the detection and quantitative analysis of DNA.
• Free pyrimidine and purine bases are easily separated by chromatographic
or electrophoretic methods.
Nucleoside
A base linked with a pentose sugar molecule is called a nucleoside, i.e., Sugar +
Base. The linkage of deoxyribose sugar with a base results in the formation of a
deoxyribonucleoside. Obviously, four different kind of deoxyribonucleosides are
found in DNA.
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DNA: Discovery, These are as follows:
Structure, Forms and
Properties • Deoxyadenosine
• Deoxyguanosine
NOTES • Deoxythymidine
• Deoxycytidine
As earlier pointed out, in a nucleoside C1, of the sugar makes β-N glycoside
bond with the N1 and N9 of the pyrimidine and purine bases, respectively. This
reaction is catalysed by synthetase enzymes and releases one —OH group from
the β position of C1 of sugar and one H atom linked the N of pyrimidine or
purine.
The combination of this -OH and H results in the formation of one molecule
of H2O:

General Properties of Nucleoside

Nucleosides show the following properties:
• Like the free bases, free nucleosides occur only in trace amounts in most
cells.
• Nucleosides are much more soluble in water than the corresponding free
bases.
• Nucleosides are relatively soluble in alkali.
• The purine nucleosides are rather readily hydrolysed by acid to yield free
base and the pentose sugar.
• The pyrimidine nucleosides are resistant to acid hydrolysis.
• Both type of nucleosides are hydrolysed by specific enzyme-nucleosides.
• They are readily separated and identified by chromatographic method.
Deoxyribonucleotide
The derivation of a nucleotide from a nucleoside occurs by the addition of one or
more molecule of phosphoric acid. When a nucleotide is derived from
deoxyribonucleoside, it is termed as deoxyribonucleotide. The deoxyribonucleotide
present in DNA consists of only one phosphate group, which is attached to the 5´
carbon of the deoxyribose sugar.
Therefore, the nucleotides are named deoxyadenylic acid or Adenosine 5´
MonoPhosphate (dAMP), deoxyguanylic acid or Guanosine 5´ MonoPhosphate
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(dGMP), deoxycytidylic acid or Deoxycytidine 5´ MonoPhosphate (dCMP) and DNA: Discovery,
Structure, Forms and
thymidylic acid or Thymidine 5´ MonoPhosphate (dTMP) (Refer Table 1.1). Properties

Table 1.1 Sugar and the Various Bases, Nucleosides and Nucleotides found in DNA
NOTES

Formation of a deoxyribonucleotide occurs as a result of linkage between a

phosphate molecule and a sugar molecule. The catalysis of this reaction is brought
about by the enzyme phosphokinase. In this reaction, out of three hydroxyl groups
of phosphate, one hydrogen atom from one hydroxyl group and one hydroxyl
group from 5´ carbon of sugar come out and they combine to form a water molecule,
i.e., one molecule water releases during this reaction.
In all deoxyribonucleotides, the phosphate and sugar parts are the same but
they only differ because of the presence of four different bases.
General Properties of Deoxyribonucleotide
• All the deoxyribonucleotide show strong ultraviolet absorption owing to the
presence of a pyrimidine or purine base.
• The phosphate groups are strong acid at pH 7.0.
• Separation of nucleotides can be easily done with the help of ion- exchange
chromatography.
Polynucleotides and its Schematic Representation
The monomeric deoxynucleotides in DNA are joined together by 3´, 5´-
phosphodiester bridges. DNA (or RNA) structure is often represented in a short-
hand form. The horizontal line shows the carbon chain of sugar with base attached
to C1. Near the middle of the horizontal line is C3– phosphate linkage while at the
other end of the line is C– phosphate linkage.

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DNA: Discovery,
Structure, Forms and
Properties

NOTES

Fig. 1.5 Structure of Polydeoxyribonucleotide Segment Held by Phosphodiester Bonds

A number of deoxyribonucleotides covalently join together to form a long, mostly

unbranched chain or polymer, i.e., deoxyribonucleotide monomer units are the
building blocks of polynucleotide chain. The successive addition of
deoxyribonucleotide units followed by their covalent linkage by phosphodiester
bridges formation between the 5´ hydroxyl group of one nucleotide and the 3´
hydroxyl group of the next occurs accompanied by the release of one molecule of
water.
Thus one by one deoxyribonucleotide joins by phosphodiester bond and,
consequently, produces dimer, trimmer, tetramer and so for in polymer, which is
known as polynucleotide. The backbone of polynucleotide chain consists of
alternating phosphates and pentoses.
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 DNA: Discovery,
Structure, Forms and
Properties

NOTES

Fig. 1.7 Nucleotides are Joined Together by a Phosphodiester Linkage between 5’ and
3’ Caron Atoms to form Nucleic Acid

The phosphoric acid utilizes two of its three hydroxyl groups in the 3´ , 5´ diester
links. The remaining third hydroxyl group in its ionic state thus makes the strongly
electronegative oxygen atom, thereby imparting polarity to the polynucleotide and
making the polynucleotide chain a highly polyanionic structure. As a result the
polynucleotide chain shows its acid properties. This free acid group also leads to
the nucleic acid being highly basophilic, i.e., they stain readily with basic dyes and
also enables the DNA of eukaryotic cell to form ionic bonds with basic histone
proteins forming a nucleoprotein complex called chromatin. A polynucleotide chain
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DNA: Discovery, has a definite direction. If it starts from carbon 3´ it would end in carbon 5´ and
Structure, Forms and
Properties if it starts from carbon 5´ it would end in carbon 3´. Sometimes it is written as
3´ → 5´ or 5´ → 3´ or 3´ prime to 5´ prime or 5´ prime to 3´ prime. A polynucleotide
sequence containing, for example, pCpGp from left to right represents p5´ C3´
NOTES p5´ G3p. The capital letters represents the base and the small p represents the
phosphate within phosphodiester bond. This bond is very important and especially
vulnerable to hydrolytic cleavage—chemically and enzymatically.
1.2.2 Properties and Characteristics of DNA
James Watson and Francis Crick proposed the double helical structure of DNA
and were awarded the Nobel Prize in 1962 for the same. The interpretation of
DNA structure proved to be a milestone in the era of modern biology. The structure
of DNA double helix is similar to a twisted ladder.
The major features of Watson-Crick Model of DNA (now known as B-
DNA) are listed below (Refer Figure 1.8).

Fig.1.8 Watson-Crick Model of DNA Helix and

Complementary Base Pairing in DNA Helix

• DNA is the largest biomolecule in the cell.

• DNA is negatively charged and dextrorotatory.
• Molecular configuration of DNA is 3D.
• Being structurally stable the chances of occurrence of any heritable changes
(mutation) are very less.
• The DNA has a right handed double helical structure which comprises of
two polydeoxyribonucleotide chains (strands) twisted or twirled around
each other on a common axis.
• The two chains or strands of DNA have antiparallel polarity, 5´ → 3´ in one
and 3´ → 5´ in other. This is comparable to two parallel adjoining roads
which carry traffic in opposite direction.
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 DNA: Discovery,
• The breadth (or diameter) of a double helix is 20 Å (2 nm). Structure, Forms and
• Each strand of DNA comprises of a backbone composed of hydrophilic Properties

deoxyribose phosphate (3´-5´ phosphodiester bonds) on the outside

(periphery) of the molecule whereas the hydrophobic bases are stacked on NOTES
the inner side (core).
• The two polynucleotide chains are complementary to each other owing to
the base pairing of nitrogen bases but are not identical to each other.
• A large sized purine always comes opposite a small sized pyrimidine. This
generates uniform distance between two strands of helix.
• Adenine (A) of one polynucleotide chain is held to Thymine (T) of opposite
chain by two hydrogen bonds. Cytosine (C) of one chain is similarly held to
guanine of the other chain by three hydrogen bonds. The G C bond is
stronger by about 50% as compared to A = T bond.
• The double chain is coiled in a helical fashion. The coiling is right handed.
This coiling produces minor and major grooves alternately.
• The pitch of helix is 3.4 nm (34 Å) with roughly 10 base pairs in each turn.
The average distance between two adjacent base pairs comes to about
0.34 nm (0.34 x 10-9 m or 3.4 Å).
• Planes of adjacent base pairs are stacked over one another. Alongwith
hydrogen bonding, the stacking confers stability to the helical structure.
• DNA is acidic. For its compaction, it requires basic (histone) proteins. The
histone proteins are +vely charged and occupy the major grooves of DNA
at an angle of 30° to helix axis.
• The hydrogen bonds are formation takes place between a purine and a
pyrimidine only. On one hand if two purines were to face each other, they
would not be able to fit into the allowable space and on the other hand two
pyrimidines would be too far apart from each other to be able to form
hydrogen bonds. Therefore the only base pair arrangement possible in DNA
structure was found to be A-T, T-A, G-C and C-G.
• Chargaff’s rule was substantiated by the complementary base pairing
occurring in DNA helix. The content of adenine was found to be equal to
that of Thymine (A = T) and that of guanine was found to be equal to that of
Cytosine (G = C).
• The template strand or sense strand, which is one of the two strands holds
the genetic information. The opposite strand is known as the antisense strand.
Along the phosphodiester backbone various major (wide) and minor
(narrow) grooves are present. These grooves facilitate easy interaction with
the proteins without causing any disruption in base pairs and the double
helix.
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DNA: Discovery, • Due to the ability of double helix structure of DNA to maintain two copies
Structure, Forms and
Properties of the information at all times, any information that may be lost due to errors
in replication or by DNA damage is often retrievable from an undamaged
complementary strand.
NOTES • Hydrogen bonds which hold the two strands of DNA helix together, if
disrupted due to changes in pH or increase in temperature lead to the
separation of the polynucleotide strands. This phenomenon involving the
loss of helical structure of DNA is called as denaturation or melting. However
the phosphodiester bonds are not broken by denaturation. Increase in
absorbance at 260 nm can be used as a measure of loss of helical structures
(in a spectrophotometer).
• Since A-T base pair has only 2H bonds, the area rich in A-T base pairs can
undergo easy denaturation (melting). These areas are called low melting
areas because they denature at comparatively low temperature. The area
rich in G- C base pairs (called high melting area) is comparatively more
stable and dense because of the presence of three hydrogen bonds
connecting the G-C bases.
• These areas have high temperature of melting (Tm). On melting the viscosity
of DNA decreases. The denatured DNA has the tendency to reassociate,
i.e., the DNA strands separated by melting at 82-90°C can reassociate
and form duplex on cooling to temperature at 65°C. It is called renaturation
or annealing. Renaturation (or reannealing) is the process in which the
separated complementary DNA strands can form a double helix.
• It has been observed that denatured or separated DNA strands absorb
more light energy in comparison to the intact DNA double strand. This
increased absorption of light energy by separated or denatured DNA strands
is known as hyperchromatic effect. This effect aids in differentiating between
single or double stranded DNA.
• Melting temperature (Tm) is the temperature at which half of the helical
structure of DNA is lost. Owing to the higher stability of G-C base (due to
3 hydrogen bonds) in comparison to A-T base pairs (2 hydrogen bonds),
the Tm for DNAs with higher G-C content is greater.
• Thus, the Tm for 35% G-C content is 65°C whereas for 50% G-C content
it is 70°C. Formamide leads to destabilization of hydrogen bonds of base
pairs thereby lowering the Tm and hence it is used in recombinant DNA
experiments.
• There are certain areas in DNA duplex where sequence of nucleotides is
the same but opposite in the two strands. These sequences are recognised
by restriction endonucleases and are used in genetic engineering. Given
hereunder sequence of bases in one strand (3´ → 5´ ) is GAATTC. It is
exactly the same in other strand when read in 5´ → 3´ direction. It is similar
to palindrome words having same words in both forward and backward
direction, for example NITIN, MALAYALAM.
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 DNA: Discovery,
Structure, Forms and
Properties

NOTES
Repetitive DNA, as the name suggests is the DNA having multiple copies of identical
sequences of nitrogen bases.
• The number of copies of the same base sequence varies from a few to
millions.
• DNA possessing a single copy of base sequences is known as a unique
DNA. It is made of functional genes. However the rRNA genes are repeated
several times.
• Repetitive DNA may be found in tandem or inter­spersed with unique
sequences.
• It is of two types, highly repetitive and moderately repetitive. Highly repetitive
DNA consists of short sequences of less than 10 base pairs which are
repeated millions of times. They occur in pericentromeric regions,
heterochromatic regions of Y-chromosomes and satellite regions. On the
other hand moderately repetitive DNA comprises of a few hundred base
pairs repeated at least 1000 times. It occurs in telomeres, centromeres and
transposons.
• The size of tandem clusters tends to be highly polymorphic, with wide
variations between individuals because tandemly repeated sequences are
more liable to undergo misalignments during chromosome pairing. These
smaller clusters of such sequences find use in the technique of ‘DNA-finger-
printing’ as they can be used to characterize individual genomes.
• Satellite DNA is a part of repetitive DNA which has long repetitive nucleotide
sequences in tandem that forms a separate fraction on density
ultracentrifugation. Depending upon the number of base pairs involved in
repeat regions, satellite DNA is of two types namely microsatellite sequences
(1-6 bp repeat units flanked by conserved sequences) and minisatellite
sequences (11-60 bp flanked by conserved restriction sites). The latter are
hyper variable and are specific for each individual. They are being used for
DNA matching or finger printing as first found out by Jeffreys et al, (1985).
• DNA has two types of segments namely coding and non-coding on the
basis of whether the segment can form a functional product or not.
• In Eukaryotes most of the DNA is constituted by non-coding segments as
most of the segments do not form any functional product. They often possess
repeated sequences or repetitive DNA. Most of them have fixed positions.
• Some can move from one place to another and hence these mobile sequences
are termed as jumping genes or transposons. In prokaryotes noncoding or
non-functional DNA is present in small amounts.
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DNA: Discovery, • Coding DNA consists of coding DNA sequences which are of 2 types,
Structure, Forms and
Properties viz., protein coding sequences coding for all proteins except histone and
non-protein coding sequences for tRNA, rRNA and histones.

NOTES Properties of DNA

Following are some properties of DNA:
Denaturation of DNA
Hydrogen bonds which hold the two strands of DNA helix together, if disrupted
due to changes in pH or increase in temperature lead to the separation of the
polynucleotide strands. This phenomenon involving the loss of helical structure of
DNA is called as denaturation or melting. However, the phosphodiester bonds
are not broken by denaturation. Melting temperature (Tm) is the temperature at
which half of the helical structure of DNA is lost. Owing to the higher stability of
G-C base (due to 3 hydrogen bonds) in comparison to A-T base pairs (2 hydrogen
bonds), the Tm for DNAs with higher G-C content is greater.
Denaturation of DNA can be due to several factors:
• Temperature: Heating up of a solution of DNA at a temperature of 90°C
or above leads to the separation of double stranded DNA molecule into
single strands. This property of DNA is often used for setting up a
polymerase chain reaction. In a DNA molecule, A-T base pair has only 2H
bonds, the area rich in A-T base pairs can undergo easy denaturation
(melting). These areas are called low melting areas because they denature
at comparatively low temperature. The area rich in G- C base pairs (called
high melting area) is comparatively more stable and dense because of the
presence of three hydrogen bonds connecting the G-C bases. These areas
have high temperature of melting (Tm). Thus, the Tm for 35% G-C content
is 65°C whereas for 50% G-C content it is 70°C.
• By the Action of Chemical Agents: Chemical agents, such as urea and
formamide can also cause denaturation of DNA molecule by enhancing the
solubility of the purine and pyrimidine groups.
• By the Action of pH: An acidic pH value of 2-3 or an alkaline pH value of
12 can bring about the denaturation of DNA.
Hyperchromicity
It has been observed that denatured or separated DNA strands at 260nm absorb
more light energy due to unstacking of base pairs in comparison to the intact DNA
double strand. This increased absorption of light energy by separated or denatured
DNA strands is known as hyperchromatic effect. This effect aids in differentiating
between single or double stranded DNA. As the plot shows, that an elevated
temperature corresponds to the denaturation of DNA, which is further followed
by the increased absorbance of light at 260nm (Refer Figure 1.9).
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 DNA: Discovery,
Structure, Forms and
Properties

NOTES

Fig. 1.9 Correlation between Melting Curve of DNA and Hyperchromatic Effect

Renaturation of DNA
The denatured DNA has the tendency to reassociate, i.e., the DNA strands
separated by melting at 82-90°C can reassociate and form duplex on cooling to
temperature at 65°C. It is called renaturation or annealing. Renaturation (or
reannealing) is the process in which the separated complementary DNA strands
can form a double helix.
Factors affecting the degree of renaturation (Refer Figure 1.10):
• C0: It is defined as the concentration of double stranded DNA prior to
denaturation. It is generally measured as nucleotides per unit volume.
• T: The duration period of the renaturation process in seconds.
• t1/2: Time taken for renaturation to proceed half way.
• C0t1/2 Value: A multiplication product of C0 and t1/2.
As a thumb rule, larger the C0t1/2 value greater is the complexity of the DNA. A
cot curve based on renaturation kinetics helps us to decipher the complexity of the
DNA molecule. Thus, repetitive DNA sequence having less complexity will show
low C0t1/2 value when compared to highly complex unique DNA sequences.

Fig. 1.10 COt-Curve of Human DNA

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DNA: Discovery, 1.2.3 Conformations of DNA Double Helix
Structure, Forms and
Properties
Variation in the conformation of the nucleotides of DNA occurs due to differences
resulting in conformational variants of DNA. The double helical structure of DNA
NOTES occurs in at least 6 varying forms- A to E and Z, among which, B, A and Z forms
are essential.
B-DNA
Watson and Crick found the B-form of DNA double helix to be the most prevalent
form under physiological conditions. Each turn of the B-form double helix has a
width of 2nm and consists of 10 base pairs covering a distance of 3.4 nm. In this
model the base pairs are found to lie at an angle of nearly 90 degrees to the axis of
helix. B-DNA is more hydrated and is predominantly found DNA in living cells as
it is physiologically and biologically the active form.
However it has been observed that it has a tendency to get changed and
hence can get changed into other forms. Temporary change of a right handed
DNA into the left handed at least for a short distance has been observed. Such
changes may become a cause of changes in gene expression. The diameter of the
helix is 20 Å. The pitch, i.e., the length of helix needed to complete one turn, is 34
Å. The distance between two base pairs is 3.4 Å. In the physiologic solution,
there are about 10.5 base pairs in one pitch rather than 10.0 found in fibre. The
axial rise of helix per base pair is 3.37 Å. The tilt of a base pair is 6.3 Å.
Conversion of B-form DNA to other different forms of DNA occurs because of
lowering of water activity, for example by addition of ethanol, by a low humidity,
or by a high salt concentration.
A-DNA (Alternate DNA)
The A-structure is induced in DNA in 70% to 75% ethanol and is found in fibres
of DNA in a dehydrated state. The primary difference between A and B helices
lies in the sugar ring conformation (pucker). The sugars are C´ 3-endo in the A-
form but C´ 2 endo in the B-form.
It is also a right-handed helix consisting of 11 base pairs per turn. An eminent
feature being that it has base pairs which are tilted away from the central axis by
an angle of 20° away from the central axis. The length of one pitch is 28.15 Å.
There are 11-12 base pairs per pitch. The axial rise per base pair is 2.56 Å. There
is a tilting of base pairs from the axis of the helix of 20.2. The A-structure is
induced in DNA in 70% to 75% ethanol and is found in fibres of DNA in a
dehydrated state. The major difference between A and B helices is in the sugar
ring conformation (pucker). The sugars present at C´ 3-endo are in the A-form
but the ones present at C´ 2 endo are in the B-form. This altered sugar pucker
shortens the distance between adjacent phosphate on one strand by about 1 Å.
Thus A-DNA has about 11 and 12 base pairs per helix pitch and not 10.5 as in
case of B-form.
The second major difference in A-DNA is that the base pairs are displaced
from the central helix axis, towards the major grooves as opposed to the case of
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B-DNA wherein the base pairs are essentially centered over the helix axis. In the DNA: Discovery,
Structure, Forms and
A-structure, they are shifted by side nearly 5 Å from the centre, resulting in a Properties
ribbon-shaped helix with a cylindrical open core and a very deep but narrow
major groove. The shape of the functional groups of the bases within these grooves
undoubtedly render A and B forms clearly distinguishable by protein interacting NOTES
with the DNA. Base stacking—both intra and inter strand—is the key element in
stabilising the A helix.
C-DNA and D-DNA both are right handed but the former has 9 base pairs per
turn of spiral while the latter consists of only 8 base pairs per turn.
C-DNA
Complementary DNA (cDNA) is the DNA produced on an RNA template by
the action of reverse transcriptase (RNA-dependent DNA-polymerase). The
sequence of the cDNA becomes complementary to the RNA sequence. Unlike
RNA, DNA molecules can be cloned easily (these are called ‘cDNA clones’) by
making the cDNA double-stranded and ligated to a vector DNA. Sequence analysis
of DNA is much easier than that of RNA, thus, cDNA is the essential form in the
analysis of RNA, particularly of eukaryotic mRNA.
The size of helix of C-form DNA is greater in comparison to A type of DNA
but is smaller in comparison to B-DNA. It is about 31 Å. There are 9.33 base
pairs per turn of the helix. There is an axial rise of base pairs of 3.03 Å with a tilting
of about 7.8°C-form is found at 66% relative humidity: in presence of Li+ (Lithium)
ions.
D-Form and E-Form DNA
D-form and E-form of the DNA are extreme variants and hence are very rarely
found. In D-form 8 base pairs per turn of helix are present. An axial rise of base
pairs is 3.03 Å with tilting of about 16.7°. In case of E-form, there are 7.5 base
pairs per turn of helix.
Z-DNA (Zigzag DNA)
Z-DNA is one of the many possible double helical structures of DNA. It is a left-
handed double helical structure in which the helix winds to the left in a zigzag
pattern (instead of to the right, like the more common B-DNA form). Z-DNA is
thought to be one of three biologically active double-helical structures along with A-
and B-DNA.
It is a left-handed helix having a zigzag backbone. Due to the somewhat
‘zigzag’ fashion in which the polynucleotide strands of DNA move, the name Z-
DNA is coined for this form. It comprises of alternate purine and pyrimidine bases
with each turn consisting of 45 Å length and 12 base pairs in addition to a single
groove. Transition between different helical forms of DNA is believed to play a
major role in regulation of gene expression.
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DNA: Discovery, Z-DNA is a new family of DNA structure, which is left-handed, i.e., the
Structure, Forms and
Properties strands of DNA are twisted towards left-side instead of the usual right-handed
twisting. B-form is twisted clockwise whereas Z-form is twisted anticlockwise.
Hence the two possible helical forms of DNA are found to be mirror images of
NOTES each other. The Z-form of DNA was discovered by Alexander Rich and his
colleagues of USA. Soon the left-handed DNA was observed in other lab­oratories,
but only under certain conditions. Factors promoting the Z-structure comprise of
methylation, bromination, specific DNA binding protein and sufficient torsional
stress as in negatively supercoiled DNA. Due to zigzag course of the backbones
the Z-DNA was so named.
In addition to the left-handedness of Z-DNA, it is the orientation of the
glycosyl bonds which makes it different from the A and B forms. The syn and anti-
orientations alternate, unlike in the case of the all the anti conformation in A- and
B- DNA. The pyrimidine nucleotides are in the standard anti conformation with a
C´2-endo sugar pucker, while the purine residues are syn and contain a
C´3-endo conformation. The Z-form is more likely to occur in alternating purine-
pyrimidine sequences because steric repulsion makes it less-favourable for a
pyrimidine to adopt the syn conformation. The crystal structures have a strong
dinucleotide repeat unit due to the large alternation in helix twist between the
C and G in a CpG step, which is about 15° while that between the G and C in the
subsequent GpC is close to 45°. Two types of grooves, viz., one major and one
minor are present in both B-DNA and A-DNA. In contrast to this the Z- DNA
possesses only a single groove, which is quite deep, extending to the axis of the
helix. The diameter of Z-DNA is about 18 Å and the length of pith is 45 Å. The
length of one pitch of Z-DNA is 45 Å. Number of base pairs/pitch is 12. The axial
rise per base in Z-DNA is 3.7 Å. Tilting of base pairs from the axis of helix is 7°.
Diameter of the helix is 18 Å. Z-DNA is currently a matter of great interest. It also
occurs in nature. Its presence has also been established in the chromosome of the
fruit-fly Drosophila melanogaster and also in the chromosomes of some other
organisms. There is growing evidence that certain DNA regions rich in Guanine
and Cytosine within long molecules do adopt a Z-like conformation. This unique
form of DNA is presumed to have a role in the regulation of gene activity.
It could act as a recognition signal for some important function of DNA. To
understand the function of Z-DNA in fruit fly or Drosophila melanogaster
chromosomes, research on synthetic DNA was conducted. It has been
demonstrated that purine-pyrimidine sequences can undergo transitions between
right- handed B-conformation depending on the salt concentration or on chemical
modification of the bases. This reversibility indicates that Z- DNA has some
regulatory role. For example, on methylation the establishment of some nucleotide
sequences in the Z-form occurs. Methylation and demethylation are very important
in controlling the activity of genes. There could be many possibilities by which the
Z- DNA controls the gene regulations, such as when certain control sites are
established in the Z-form by methylation—regulatory protein binds to the site and
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keeps gene turned off; demethylation might switch the site to the B- conformation, DNA: Discovery,
Structure, Forms and
causing the regulatory molecule to let go. Properties
The basic helix geometry of Z-DNA is:
• The length of one pitch is 45 Å. NOTES
• Number of base pairs/pitch is 12.
• The axial rise per base is 3.7 Å.
• Tilting of base pairs from the axis of helix is 7°.
• Diameter of the helix is 18 Å.
Identification of Z-DNA
Rich et al. could raised antibodies by injecting rabbits with short, brominated
double helices of alternating G and C nucleotides of Z-DNA. The rabbit made
antibodies that were highly specific for Z-DNA and did not react at all with the B-
form.
Purified rabbit antibodies for Z-DNA were used to probe biological materials
for the pres­ence of Z-DNA. The polytene chromosome of D. melanogaster were
treated with rabbit’s anti z-antibody and incubated to enable the antibody to bind
to any Z-DNA in the chromosomes.
The unbound antibodies were washed away. Further, the bound antibodies
were made visible by adding a goat antibody specific for rabbit one. The goat
antibody was conjugated to a fluorescent dye, so that it glowed under ultraviolet
illumination. The anti z-antibody could be seen to bind to the chromosomes in a
distinct and reproducible segmented pattern (Refer Figure 1.11).

Fig. 1.11 Identification of Z-DNA

By comparing ultraviolet micrographs that reveal the bands and inter-bands of

polytene chromosomes, it was established that the fluorescent segments are the
inter-bands and not the bands. Therefore, there must be Z-DNA in the inter-
bands.
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DNA: Discovery, It is conceivable that some Z-DNA is also present in the bands but is not
Structure, Forms and
Properties visibly stained by the antibodies. Presently it appears that Z-DNA is either more
abundant or more accessible in the inter bands than the bands.
NOTES Resemblances and Differences between B-DNA and Z-DNA
• Both B-DNA and Z-DNA have a double helical structure.
• The strands of both B-DNA and Z-DNA are antiparallel.
• G C pairing is found in both B-DNA and Z-DNA.
• B-DNA is right-handed whereas Z-DNA is left-handed.
·While sugar-phosphate backbone in B-DNA is has a regular structure, the structure
of this sugar-phosphate backbone in Z-DNA follows zigzag course.
• In B-DNA one complete helix is 34 A while in Z-DNA it is 45 Å.
• In Z-DNA, opposite orientations are exhibited by adjacent sugar residues
whereas in B-DNA they exhibit the same orientation. This leads to the
presence of dinucleotide units in Z-DNA as opposed to the mononucleotide
units present in B-DNA
• In B-DNA there are ten base pairs at each turn while there are twelve base
pairs at each turn of Z-DNA.
• The diameter of B-DNA is 20 Å whereas it is 18 Å in Z-DNA.
• The bases in Z-DNA are closer to the axis as compared to the bases in
B-DNA.
• In B- DNA the glycosidic torsion angle is anti whereas in B-DNA it is syn.
• Sugar pucker for B-DNA is C´2 endo and for Z-DNA is C´3 endo in
deoxyguanosine.
• The angle of twist per repeating unit, i.e., dinucleotide is 60° in Z-DNA in
contrast to 36° in B- DNA.
• In B-DNA the base pair tilt is 6° whereas in Z-DNA it is 7°.
Types of DNA in Different Organisms
Various studies on DNA of viruses, bacteria and eukaryotes have lead to a number
of surpris­ing findings. One of the findings being that viral and bacterial DNAs are
very simple. Molecular weight is found to be very low and approximate number of
gene is found to be relatively few. DNA being in no association with any proteins,
i.e., a naked DNA thread, is often referred to as genophore.
Second is that eukaryotic DNA is very complex. Molecular weight is
comparatively high and the DNA contains several genes. It always makes a complex
with basic histone protein.
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 Besides this, a number of variations still exist with respect to the structure of DNA: Discovery,
Structure, Forms and
DNA that serves as genome material in viruses, bacteria and Eukaryotic organisms. Properties
Viral DNA
Viruses comprises of either DNA or genetic RNA as genetic material but never NOTES
both. While some of the viral DNA are Double- Stranded (DS) as in prokaryotic
and Eukaryotic cell, others are Single-Stranded (SS). Among them some are linear
while others are circular. Hence a number of possibilities exist with respect to
structure of DNA.
The DNAs of lamda (λ) bacteriophages and ϕ x 174 have received much
attention. The DNA of lamda (λ) bacteriophage is a linear duplex DNA having a
molecular weight of 32 million. The DNA’ of ϕ x 174 virus, which is one of the
smallest DNA viruses known is circular and single-stranded with molecular weight
of 1.7 million.
Robert Sinsheimer in 1959 made two important observations on the DNA
of 0 x 174. First the ratio of A/T and G/C were not one as is followed by the rules
of base pairing in the double helix. Second, the amino groups of the purine and
pyrimidine rings reacted readily with formaldehyde—a property that is generally
seen only with RNA or heat denatured DNA.
On the basis of such observation Sinsheimer concluded that ϕ x 174 contains
a single DNA strand. Electron microscopic studies indicate the circularity of 0 x
174 DNA. The next step in confirming the circularity of ϕ x 174 is that exonucleases
are unable to use the intact DNA as a substrate because of a lack of free ends.
This DNA has approximately 5,375 deoxyribonucleotides making up nine genes
in the circle out of which some genes are overlapping.
The linear viral DNAs possess certain character­istic features. For example,
the linear duplex DNA of A phage has cohesive or sticky ends. The 5´ ends of
these duplex DNAs project as single-strands beyond 3´ ends. Twelve
deoxyribonucleotides on these ends are complementary arid thus pair to form
circular structure which can then be covalently linked by DNA ligase.
Sedimentation studies on polyoma DNA show that it may occur in three
forms. A supercoiled conformation, resulting from under-winding during synthesis,
can be relaxed to a simple circular form if one strand of duplex DNA is severed. If
both strands are severed sufficiently close together it leads to a linear molecule
formation.
The liner single-stranded DNA (for example, Provirus) consists of certain
hairpin-like regions at each of its end. Besides this, ssDNA is completely devoid
of random coil, base stacking, intra-strand hydrogen bonding and other types of
pairing. Different forms of viral DNA molecules found in a variety of viruses are
shown briefly in Figure 1.12.
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DNA: Discovery,
Structure, Forms and
Properties

NOTES

Fig. 1.12 Forms of DNA Molecules found in a Variety of Viruses

Bacterial DNA
The bacterial DNA that is huge in size as compared to that present in viruses. The
DNA of Escherichia coli appears to consist of a single, enormous double-stranded
DNA molecule with a molecular weight of about 2.8 x 109, a thickness of about
2.0 nm and a contour length of about 1,360 µm. Escherichia coli DNA contains
4 million deoxyribonucleotide pairs.
This DNA is a closed and unfolded circle. Due to interaction with nucleoid
proteins and RNA, the circular DNA folds into a number of loops. The loops
being of limited size have around 100 per genome. Each loop is independently
supercoiled.
Evidence in support of this comes from studies where nucleoid proteins are
dissociated or denatured or nucleoids are subjected to RNAase treatment. In
both these cases, the DNA loses its supercoiled state and unfolds completely on
completion of RNA hydrolysis. Thus nucleoid proteins and RNA appear to stabilize
the supercoiled state.
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 In addition to the main DNA present in the nucleoid, the cytoplasm of most DNA: Discovery,
Structure, Forms and
bacterial cell contains 1 to 20 mini circular, extra chromosomal, self-replicating, Properties
duplex DNA. These are called plasmids. The size of the plasmid lies between 5 to
100 mega Dalton.
NOTES
Many different plasmids have the ability to integrate with the host chromosome
and a plasmid possessing this ability is known as episome. Transfer of plasmids
from one bacterial cell to another may also occur during conjugation.
Plasmids contain sufficient genetic information for their own replication.
Plasmids also have certain specific properties like antibiotic and heavy metal
resistance, nitrogen fixation, pollutant degradation, bacteriocin and toxin production,
etc.
The organisation of DNA in prokaryotes differs from that in eukaryotes in
several major respects. At the gross level, Eukaryotic DNA is generally linear
whereas Prokaryotic DNA is circular. Eukaryotic DNAs are distributed in a number
of chromosomes rather than one.
Furthermore, in prokaryotic DNA, protein coding genes are not split,
whereas eukaryotic protein coding genes are split and the coding ‘exons’ are
interspersed with generally non coding ‘introns’. Unlike prokaryotes, eukaryotes
have three types of RNA polymerase each of which is responsible for the
transcription of different classes of genes.
Unlike Eukaryotes, almost all regulation of Prokaryotes is at the level of
transcription, in most cases translation of the mRNA commences before
transcription is completed. Furthermore, and unlike Eukaryotes genes encoding
enzymes forming part of a common biochemical pathway are often clustered
together and coordinately regulated in operons.
The clustering of genes in this ordered manner, therefore, requires only a
single regulatory switch for coordinate expression.
Eukaryotic Nuclear DNA
The nuclear DNA of the eukaryotic cell is present in the nucleoplasm of a nucleus
which is surrounded by a membrane. It is neither singular nor circular like bacteria.
Additionally it is always linear and double-stranded. Combination of single DNA
duplex with the basic protein results in the formation of a complex called histone.
This nucleoprotein complex is known as chromatin which is the unit of
genome. The condensed form of chromatin is called as chromosome. The nucleus
of Eukaryotic cells may contain few or many chromosomes, depending on the
species. The size of chromosome varies from species to species which implies that
size of the DNA molecule is also variable. The largest chromosome of Drosophila,
however, contains a DNAmolecule of about 4.0 cm long having a molecular weight
of 80 x 109, which is nearly 40 times larger than the DNA of Escherichia coli.
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DNA: Discovery, The DNA of the Eukaryotic cells undergoes folding and supercoiling many
Structure, Forms and
Properties times to accommodate themselves within the chromosomes. The physical forms
of chromosomes also vary according to the cell cycle. During interphase, they are
extended or uncoiled.
NOTES
During prophase, chromosomes coil and shorten until they finally reach the
metaphase. During telophase they begin to uncoil and again attain the relaxed
condition when the interphase of the next all cycle is reached.
Therefore, change in physical form of chromosome during cell cycle may
bring about some additional changes in the folding and supercoiling pattern of
DNA that is present inside the chromosome. It has been calculated that human
chromosome No. 13 contains a DNA molecule about 32,000 µm long. This DNA
undergoes looping and supercoiling to form a chromatid about 6 µm long and 0.8
µm in diameter.
The chromatin fibres of somatic cells are about 20 nm in diameter. In addition
to DNA histone complex, chromatin also contains some acidic non-histone proteins,
some enzyme proteins like DNA polymerase, as well as nuclear RNA and some
lipids.
Rat liver chromatin has been used as a model for chromatin. It possesses a
histone to DNA ratio near 1: 1, a non-histone protein to DNA ratio of 0.6:1 and
RNA/DNA ratio of 0.1:1.
Eukaryotic Non-Chromosomal DNA
In contrast to popular earlier believe that the DNA of the eukaryotic cell only
resides in the chromosomes was proved wrong when with the refinement of
methodology and improvement of microscopy, it was demonstrated quite
convincingly that in certain Eukaryotes, DNA is present in certain organ cells,
such as mitochondria, plastids, centrioles and the yolk spherules of the egg.
Generally, such DNAs are called cytoplasmic or non-chromosomal or extra
chromosomal DNA to distinguish them from nuclear DNA. These DNAs are
unique to the organelle. Their structure and properties of such DNA are quite
distinct from that of nuclear DNA. These extra-nuclear DNA molecules possess
the capability of partial self-replication and being circular they bear a resemblance
with the DNA of virus and bacteria.
Mitochondrial DNA: Mitochondria of Eukaryotic cells contain DNA. It is always
double-stranded, circular and not associated with histone or any other proteins.
This DNA has some other names, such as mt DNA or chondriome. There are
about four to six identical copies of DNA per mitochondrion. The DNA exists
within the mitochondrial matrix and apparently attaches at the point of inner
membrane of mitochondrion.
The fact that all the mt DNAs in a single organism are identical was strongly
supported by the result of DNA-DNA renaturation and restriction endonuclease
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studies. In animals the molecular weight ranges between 10 x 106 Dalton whereas DNA: Discovery,
Structure, Forms and
in higher plants it lies between 70 x 106 Daltons. Properties
The green algae, Chlamydomonas has a linear mitochondrial genome of
only 16,000 nucleotides pairs, the same size as in animals. The mitochondrial NOTES
DNA of Saccharomyces cerevisiae (yeast) have been sequenced and only about
one-third of them code for protein.
This finding raises the possibility that the remaining part of mt DNA of yeast
contains junk DNA. In human cells, both strands of the mitochondrial DNA are
transcribed at the same time from a single promoter region on each strand.
The transcripts made on one strand is called the heavy strand or H-strand
because it yields huge amount of RNAs including two rRNAs, most of the tRNAs
and one small Poly- A containing RNA. In contrast, the light strand (L-strand)
produces only eight tRNAs and one small ploy A containing RNA.
Unlike human mitochondrial genomes, plant mitochondrial genomes contain
introns. In yeasts the same mitochondrial gene may have an intron in one strain but
in some other strains such introns are absent. The optional introns that are present
in few yeast strains are able to move in and out of genomes like transposable
elements.
Apparently during replication of mitochondrial DNA the formation of certain
interlocked circle like forms of DNA by small fraction of the mitochondrial DNA
has been observed. These forms came to be known as catenated forms or dimers.
The role of mt DNA in the mitochondrion is similar to the role of nuclear
DNA. But mt DNA cannot be expressed without assistance from the nucleus. In
fact, it cannot even be replicated without, such assistance. This is the level at
which the molecular dependency of the mitochondrion on the nuclear genetic material
is observed.
Chloroplast DNA: Chloroplasts, like mitochondria, have their own DNA (ct
DNA). This DNA is generally present in multiple copies with as many as 20 to 60
ct DNAs per chloroplast. The molecular weight of ct DNA ranges from 85 x
106 Daltons. The length of ct DNA ranges from 40 to 60 µm. Isolated ct DNA
typically exists in a variety of forms.
Two types of dimers are found:
• Circular Dimers: Which are formed by recombination between two
monomers. It constitutes up to 10% of ct DNA.
• Catenated Dimers: In which monomers interlock like links in a chain. It
constitutes up to 2.5%. The monomers often appear as open chain duplex
in vitro, but in situ supercoiled form is preponderate. Chloroplast DNA is
made of both unique and repetitive sequences.
Yolk Spherules DNA: In sea-urchin, DNA has been isolated from the yolk
spherules. The analysis shows that the yolk contains the same amount of DNA as
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DNA: Discovery, the mitochondria. The DNA particles in yolk are generally considered as inert
Structure, Forms and
Properties storage structures.
However, there is evidence that yolk spherules are derived from mitochondria
during oogenesis. Therefore it is expected that, with the transformation of
NOTES
mitochondria into yolk spherules, it may acquire the DNA from mitochondria. But
still further experimental investigation is needed on yolk spherules DNA.
Centriolar DNA: The centrioles of the animal cells also contains DNA, which is
called centriolar DNA. This evidence comes from the studies by Randal and
Disbrey on the ciliated Paramecium by means of fluorescence microscopy of
acridine orange and autoradiography of tritiated thymidine incorporation followed
by the treatment with DNAase.
The amount of DNA per centriole is about 2 x 106 gms. The DNA in centriole
is not detectable shortly after cell division—possibly because it is too dispersed at
the time.
Other Types of DNA Structure
Apart from the normal double helical structure DNA was found to exist in certain
unusual structures which were considered to be important for molecular recognition
of DNA by various proteins and enzymes. In addition this feature was found to be
a requirement for the DNA to perform its functions in an appropriate manner.
Some of these unusual structures of DNA are briefly described below.
Bent DNA: Usually the Adenine base containing DNA tracts are found to be
rigid and straight. Bent conformation of DNA results due to the replacement of the
A-tracts by other bases or when a collapse of the helix occurs into the minor
groove of A- tract. Additionally photochemical damage or wrong pairing of bases
has been reported as the cause of bending in DNA. Certain antitumor drugs (for
example, Cisplatin) manufacture bent structure in DNA which in turn aid by taking
up proteins that cause damage to the DNA.
Triple-Stranded DNA: Additional hydrogen bonds between the bases often lead
to formation of triple-stranded DNA. Thus, the formation of a T-A-T bond can
occur when a Thymine selectively forms two Hoogsteen hydrogen bonds with the
Adenine of A-T pair. Similarly, a C+-G-C can be formed when a protonated
Cytosine forms two hydrogen bonds with Guanine of G-C pairs.
Owing to the fact that the three negatively charged backbone strands present
in the triple helix leads to an increased electrostatic repulsion, triple helical structure
is less stable than double helical structure.
Four-Stranded DNA: Four-stranded DNA or more commonly known as G-
quartets are novel tetrameric structures which are formed by polynucleotides which
have a very high content of Guanine. These tetrameric structures are planar,
connected via Hoogsteen hydrogen bonds.
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1.2.4 Forms of DNA DNA: Discovery,
Structure, Forms and
Properties
The different forms of DNA are as follows.
Heteroduplex DNA
NOTES
A heteroduplex is a double-stranded DNA molecule produced by the genetic
recombination of single complementary strands derived from two different sources,
i.e., from different homologous chromosomes or sometimes from
different organisms. For example, such heteroduplex DNA strand are formed
in hybridization processes for biochemistry-based phylogenetic analyses.
In meiosis, the process of crossing-over occurs between non-sister
chromatids, which results in new allelic combinations in the gametes. In crossing-
over, a Spo11 enzyme makes staggered nicks in a pair of sister chromatid strands
(in a tetrad organization of prophase). Subsequent enzymes trim back the 5' ends
of the strand and a protein complex binds to the 3' single-stranded ends. Rad51
protein is recruited and binds in a protein complex to search for a complementary
sequence analogous to double-strand-break repair. The filament searches for the
homologous chromosome, strand invasion occurs where the new chromosome
forms a D-loop over the bottom sister chromatid, then the ends are annealed. This
process can yield double Holliday junctions that when cut in a transversal pattern
by endonucleases form 2 heteroduplex strand products (Refer Figure 1.13).
Heteroduplex DNA is also a source of small RNAs (smRNAs), causing
post-transcriptional gene silencing.

Fig. 1.13 Heteroduplex DNA

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DNA: Discovery, The above Figure 1.13 shows heteroduplex DNA in which (A) illustrtes
Structure, Forms and
Properties heteroduplex DNA formation during denaturation and annealing. Dark green bars
represent four DNA strands (a–d) in cells harboring monoallelic mutations (orange
box). After denaturation and annealing, two types of homoduplex DNA and two
NOTES types of heteroduplex DNA were formed. (B) Identification of heteroduplex DNA
fragments by 15% PAGE assay. Since heteroduplex DNA migrates slower due to
formation of an open angle between matched and unmatched genomic regions,
homoduplex DNA and heteroduplex DNA are easily identified based on their
mobility rate.
Circular DNA
Circular DNA is DNA that forms a closed loop like structure and has no ends.
For example, mitochondrial DNA, Plasmids, Chloroplast DNA. Any of the
covalently closed DNA molecules found in bacteria, many viruses, mitochondria,
plastids, and plasmids. Small, polydisperse circular DNA’s have also been observed
in a number of eukaryotic organisms and are suggested to have homology with
chromosomal DNA and the capacity to be inserted into, and excised from,
chromosomal DNA. It is a fragment of DNA formed by a process of looping out
and deletion, containing a constant region of the mu heavy chain and the 3'-part of
the mu switch region. Circular DNA is a normal product of rearrangement among
gene segments encoding the variable regions of immunoglobulin light and heavy
chains, as well as the T-cell receptor (Refer Figure 1.14).

Fig. 1.14 Circular DNA

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Supercoiled DNA DNA: Discovery,
Structure, Forms and
Properties
When the DNA molecule contains the same number of base pairs per helical turn
it is referred to be in the relaxed state. However, if both the ends of the DNA
molecule are held rigidly such that it causes over twisting or under twisting of DNA NOTES
molecule, then such DNA is referred to as supercoiled DNA. Over twisting leads
to positive supercoiling, while under twisting leads to negative supercoiling.
Topoisomerases is a group of enzyme that removes both negative as well as positive
supercoils from DNA. This reaction requires energy, which is obtained via hydrolysis
of water. Topoisomerase present in eukaryotes and T-Phage convert the
supercoiled DNA into a relaxed DNA whereas topoisomerases named DNA
gyrase has the capability to relax negatively supercoiled DNAs (Refer Figure 1.15).

Fig. 1.15 Supercoiling of DNA, Twisting of the DNA Axis upon Itself

Functions of DNA
• Genetic Information (Genetic Blue Print): DNA being the genetic
material is the carrier or transporter of all the hereditary information, which
is coded in the arrangement of its nitrogen bases. It has the potential to
carry all types of necessary biological information and is involved in gene
action which—through a series of chemical reactions results in the ultimate
expression of characteristics within the organism. The latter property is known
as hetero-catalysis.
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DNA: Discovery,
Structure, Forms and
• Replication: DNA possesses the unique ability to produce its replica and
Properties hence produce its carbon copies (Autocatalytic function), which is
considered to be a very important property of DNA as it ensures the transfer
NOTES of genetic information from one cell to its daughters and from one generation
to the next.
• Chromosomes: The occurrence of DNA inside chromosomes is very
crucial to ensure equitable distribution of DNA during cell division.
• Recombination: During meiosis, crossing over gives rise to new
combination of genes and this process is termed as recombination. It is
crucial for most pathways of damage repair, for replication of the ends of
some linear molecules and for the initiation of replication. It provides for the
communication between DNA molecules in a population, across species
and even across kingdom boundaries, affording a means for maintaining
both genetic stability and diversity.
• Mutations: Changes in sequence of nitrogen bases caused as a result of
addition, deletion or wrong replication leads to mutations which are a major
cause or fountain head of all variations and evolution.
• Transcription: Transcription is a process through which DNA gives rise to
RNAs. It is heterocatalytic activity of DNA.
• Cellular Metabolism: It controls the metabolic reactions of the cells with
the aid of specific RNAs, synthesis of specific proteins, enzymes and
hormones.
• Differentiation: Differences in shape, size and functions of different parts
of the organism arise due to the differential functioning of certain specific
regions of DNA or genes.
• Development: The development of an organism is controlled by DNA
controls via working of an internal genetic clock with or without the help of
extrinsic information.
• DNA Finger Printing: Each individual possesses unique hypervariable
microsatellite DNA sequences which are of immense use in identification of
individuals and deciphering of their relationships. This mechanism is known
as DNA finger printing.
• Gene Therapy: Gene therapy involves rectification of defective heredity
by a process of incorporation of correct genes in place of defective ones.

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 • Antisense Therapy: Excess availability of anti-mRNA or antisense RNAs DNA: Discovery,
Structure, Forms and
will not allow the pathogenic genes to express themselves. Failure of Properties
angioplasty has been checked with the aid of this technique. A modification
of this technique is RNA interference (RNAi).
NOTES

Check Your Progress

1. What is DNA?
2. What does chemical analyses of purified DNA show?
3. Of what pyrimidine bases are composed?
4. Define purine.
5. List some general properties of pyrimidines and purines.
6. What are the four different kind of deoxyribonucleosides found in DNA?
7. Explain cDNA.

1.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. DNA is the chemical name for the molecule that carries genetic instructions
in all living things. The DNA molecule consists of two strands that wind
around one another to form a shape known as a double helix. Each strand
has a backbone made of alternating sugar (deoxyribose) and phosphate
groups. Attached to each sugar is one of four bases: Adenine (A), Cytosine
(C), Guanine (G), and Thymine (T). The two strands are held together by
bonds between the bases; Adenine bonds with Thymine, and Cytosine bonds
with Guanine. The sequence of the bases along the backbones serves as
instructions for assembling protein and RNA molecules.
2. Chemical analysis of highly purified DNA has shown that it is made of four
kinds of monomeric building blocks, each of which contains three types of
molecules:
• Phosphoric Acid
• Pentose Sugar
• Organic Bases
3. Pyrimidine bases are composed of a six-membered pyrimidine ring, which
is similar in structure to the benzene ring except that it contains nitrogen in
place of carbon at the positions of 1 and 3. Three pyrimidine derivatives are
Uracil, Thymine and Cytosine.

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DNA: Discovery, 4. Purine is a derivative of pyrimidine which comprises of a pyrimidine ring
Structure, Forms and
Properties and a five-membered imidazole ring (having nitrogen at 7 and 9 positions)
which are fused together at 5 and 4 position.
NOTES 5. All pyrimidines and purines have some general properties:
• Free pyrimidine and purine bases are relatively insoluble in water.
• Free pyrimidine and purine occur only in trace amounts in most cells.
• They are weakly basic compounds that may exist in two or more
tautomeric forms depending upon the pH.
• They strongly absorb ultraviolet light in the region 250 to 260 nm. The
alternating double and single bonds between the carbon atoms in the
nitrogenous bases can interchange continuously, producing resonance.
As a result, the bases absorb ultraviolet light at 260 nm. This property is
very useful in the detection and quantitative analysis of DNA.
• Free pyrimidine and purine bases are easily separated by
chromatographic or electrophoretic methods.
6. Four different kind of deoxyribonucleosides are found in DNA are as follows:
• Deoxyadenosine
• Deoxyguanosine
• Deoxythymidine
• Deoxycytidine
7. Complementary DNA (cDNA) is the DNA produced on an
RNA template by the action of reverse transcriptase (RNA-dependent
DNA-polymerase). The sequence of the cDNA becomes complementary
to the RNA sequence. Unlike RNA, DNA molecules can be cloned easily
(these are called ‘cDNA clones’) by making the cDNA double-stranded
and ligated to a vector DNA. Sequence analysis of DNA is much easier
than that of RNA, thus, cDNA is the essential form in the analysis of RNA,
particularly of eukaryotic mRNA.

1.4 SUMMARY

• DNA is the chemical name for the molecule that carries genetic instructions
in all living things.
• The DNA molecule consists of two strands that wind around one another
to form a shape known as a double helix.
• Each strand of DNA has a backbone made of alternating sugar (deoxyribose)
and phosphate groups. Attached to each sugar is one of four bases: Adenine
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 DNA: Discovery,
(A), Cytosine (C), Guanine (G), and Thymine (T). Structure, Forms and
Properties
• The two strands of DNA are held together by bonds between the bases;
Adenine bonds with Thymine, and Cytosine bonds with Guanine.
NOTES
• The sequence of the bases along the backbones serves as instructions for
assembling protein and RNA molecules.
• Nucleic acids are of two types - DeoxyriboNucleic Acid (DNA) and
RiboNucleic Acid (RNA), both of which primarily serve as reservoir and
transmitters of genetic information.
• Johann Friedrich Miescher, a Swiss researcher, discovered DNA in 1869.
Avery, Macleod and McCarty first demonstrated in 1944 that DNA
contained genetic information.
• Friedrich Miescher (1869) first isolated nucleic acids from pus cells. Initially
named nuclein, Hertwig (1884) believed them to be the carrier of hereditary
traits.
• The presence of purine and pyrimidine bases in nucleic acids was discovered
by Fisher (1880s).
• Deoxyribose nucleic acid was found to contain phosphoric acid as well as
deoxyribose sugar by Levene (1910) who also gave the characterization of
4 types of nucleotides present in DNA.
• In 1950, Chargaff found the content of purine and pyrimidine present in
DNA to be equal.
• One turn of the helical structure was 3.4 nm comprising of 10 layers of
bases which were stacked in it.
• The double helix model was first correctly worked out by Watson and
Crick (1953) from the X-ray photographs of Wilkins and Franklin.
• Nobel Prize was awarded to Wilkins, Watson and Crick for the same in
1962. In 1953 Watson and Crick developed a 3D, molecular model of
DNA that satisfied all the minute details obtained from X-ray photographs.
• A hall mark of their proposition complementary base pairing between the
two polynucleotide chains where the complementary pairs formed between
purine of one and pyrimidine of the other chain were held together via
hydrogen bonds (A-T, C-G).
• The phosphoric acid (H3PO4) is biologically called phosphate and it was
discovered by Levene in 1910.
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DNA: Discovery, • Phosphoric acid consists of three reactive hydroxyl groups (—OH), out of
Structure, Forms and
Properties which two are involved in forming sugar phosphate backbone of both DNA
as well as RNA.
NOTES • A phosphate group binds to the 52 carbon of one and 32 carbon of the
other adjacent pentose sugar molecule to make the phosphate diester.
• Many varying kinds of heterocyclic nitrogen comprising of ring compounds
are found in the structure of DNA. They are known as simply bases because
they can combine with H+ in acidic solution.
• Pyrimidine bases are composed of a six-membered pyrimidine ring, which
is similar in structure to the benzene ring except that it contains nitrogen in
place of carbon at the positions of 1 and 3. Three pyrimidine derivatives
are Uracil, Thymine and Cytosine.
• While Cytosine and Thymine are commonly found in DNA, Cytosine and
Uracil axe found in RNA.
• Thymine is characteristically present in DNA because thymine ensures
stability of the genetic message. Otherwise retention of the uracil would
result in mispairing and mutagenesis on subsequent replication.
• Purine is a derivative of pyrimidine which comprises of a pyrimidine ring
and a five-membered imidazole ring (having nitrogen at 7 and 9 positions)
which are fused together at 5 and 4 position.
• There are two purine compounds namely Adenine (A) and Guanine (G)
which are found in the structure of DNA. Adenine has an amino group
(-NH2) at 6 position while guanine has a keto group and an amino group at,
6 and 2 positions of carbon, respectively.
• A base linked with a pentose sugar molecule is called a nucleoside, i.e.,
sugar + base.
• The linkage of deoxyribose sugar with a base results in the formation of a
deoxyribonucleoside.
• The derivation of a nucleotide from a nucleoside occurs by the addition of
one or more molecule of phosphoric acid. When a nucleotide is derived
from deoxyribonucleoside, it is termed as deoxyribonucleotide.
• The deoxyribonucleotide present in DNA consists of only one phosphate
group, which is attached to the 5´ carbon of the deoxyribose sugar.
• Formation of a deoxyribonucleotide occurs as a result of linkage between a
phosphate molecule and a sugar molecule. The catalysis of this reaction is
brought about by the enzyme phosphokinase.
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 DNA: Discovery,
• In this reaction, out of three hydroxyl groups of phosphate, one hydrogen Structure, Forms and
atom from one hydroxyl group and one hydroxyl group from 5´ carbon of Properties

sugar come out and they combine to form a water molecule, i.e., one molecule
water releases during this reaction. NOTES
• The monomeric deoxynucleotides in DNA are joined together by 3´ , 5´ -
phosphodiester bridges. DNA (or RNA) structure is often represented in a
short-hand form.
• The horizontal line shows the carbon chain of sugar with base attached to
C1. Near the middle of the horizontal line is C3– phosphate linkage while at
the other end of the line is C– phosphate linkage.
• A number of deoxyribonucleotides covalently join together to form a long,
mostly unbranched chain or polymer, i.e., deoxyribonucleotide monomer
units are the building blocks of polynucleotide chain.
• The successive addition of deoxyribonucleotide units followed by their
covalent linkage by phosphodiester bridges formation between the 5´
hydroxyl group of one nucleotide and the 3´ hydroxyl group of the next
occurs accompanied by the release of one molecule of water.
• Hydrogen bonds which hold the two strands of DNA helix together, if
disrupted due to changes in pH or increase in temperature lead to the
separation of the polynucleotide strands. This phenomenon involving the
loss of helical structure of DNA is called as denaturation or melting.
• Melting temperature (Tm) is the temperature at which half of the helical
structure of DNA is lost.
• Watson and Crick found the B-form of DNA double helix to be the most
prevalent form under physiological conditions.
• B-DNA is more hydrated and is predominantly found DNA in living cells as
it is physiologically and biologically the active form.
• The DNA of Escherichia coli appears to consist of a single, enormous
double-stranded DNA molecule with a molecular weight of about 2.8 x
109, a thickness of about 2.0 nm and a contour length of about 1,360 µm.
Escherichia coli DNA contains 4 million deoxyribonucleotide pairs.
• Plasmids contain sufficient genetic information for their own replication.
Plasmids also have certain specific properties like antibiotic and heavy metal
resistance, nitrogen fixation, pollutant degradation, bacteriocin and toxin
production, etc.
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DNA: Discovery,
Structure, Forms and
• The nuclear DNA of the eukaryotic cell is present in the nucleoplasm of a
Properties nucleus, which is surrounded by a membrane. It is neither singular nor circular
like bacteria.
NOTES • The size of chromosome varies from species to species which implies that
size of the DNA molecule is also variable.
• A heteroduplex is a double-stranded DNA molecule produced by the
genetic recombination of single complementary strands derived from two
different sources, i.e., from different homologous chromosomes or
sometimes from different organisms.
• Circular DNA is DNA that forms a closed loop like structure and has no
ends. For example- mitochondrial DNA, Plasmids, Chloroplast DNA.
• Small, polydisperse circular DNA’s have also been observed in a number
of eukaryotic organisms and are suggested to have homology with
chromosomal DNA and the capacity to be inserted into, and excised from,
chromosomal DNA.
• When the DNA molecule contains the same number of base pairs per helical
turn it is referred to be in the relaxed state.

1.5 KEY WORDS

• Purines: Purine is a derivative of pyrimidine which comprises of a pyrimidine

ring and a five-membered imidazole ring which are fused together at 5 and
4 position.
• Deoxyribonucleotide: When a nucleotide is derived from
deoxyribonucleoside, it is termed as deoxyribonucleotide.
• Transcription: Transcription is a process through which DNA gives rise to
RNAs. It is heterocatalytic activity of DNA.
• Centriolar DNA: The centrioles of the animal cells also contains DNA,
which is called centriolar DNA.
• Four-stranded DNA: Four-stranded DNA or more commonly known as
G-quartets are novel tetrameric structures which are formed by
polynucleotides which have a very high content of guanine.
• C-DNA: Complementary DNA (cDNA) is the DNA produced on an
RNA template by the action of reverse transcriptase (RNA-dependent
DNA-polymerase).
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 • Heteroduplex: A heteroduplex is a double-stranded DNA molecule DNA: Discovery,
Structure, Forms and
produced by the genetic recombination of single complementary strands Properties
derived from two different sources.

NOTES
1.6 SELF ASSESSMENT QUESTIONS AND
EXERCISES

Short Answer Questions

1. What is DNA?
2. How many bases does DNA consists of?
3. What is pentose sugar?
4. Explain with the help of diagram structure of sugars present in nucleic acids.
5. What are nucleoside? Give its general properties.
6. Explain the structure of polydeoxyribonucleotide segment held by
phosphodiester bonds with the help of diagram.
7. What are the different types of DNA in different organisms?
Long Answer Questions

1. Discuss the structure of DNA. Also draw suitable diagrams.

2. Write a note on molecular basis of DNA.
3. Briefly discuss about the organic bases. Also draw suitable diagrams to
explain them.
4. Explain with the help of table sugar and the various bases, nucleosides and
nucleotides found in DNA.
5. List the properties and characters of DNA.
6. Discuss in detail about different forms of DNA. Also draw suitable diagrams.
7. What are the functions of DNA? Explain.

1.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
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DNA: Discovery,
Structure, Forms and
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Properties Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
NOTES Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 DNA: Replication,

UNIT 2 DNA: REPLICATION, Enzymology and Types

ENZYMOLOGY AND TYPES

NOTES
Structure
2.0 Introduction
2.1 Objectives
2.2 General Introduction
2.3 DNA Replication: An Insight
2.4 Enzymology of DNA Replication
2.5 DNA Replication Mechanism: Prokaryotic and Eukaryotic
2.5.1 Types of Replication
2.6 Answers to Check Your Progress Questions
2.7 Summary
2.8 Key Words
2.9 Self Assessment Questions and Exercises
2.10 Further Readings

2.0 INTRODUCTION

In molecular biology, DNA replication is the biological process of producing two

identical replicas of DNA from one original DNA molecule. DNA replication occurs
in all living organisms acting as the basis for biological inheritance. The cell possesses
the distinctive property of division, which makes replication of DNA essential.
DNA is made up of a double helix of two complementary strands. During
replication, these strands are separated. Each strand of the original DNA molecule
then serves as a template for the production of its counterpart, a process referred
to as semiconservative replication. As a result of semiconservative replication, the
new helix will be composed of an original DNA strand as well as a newly synthesized
strand. Cellular proofreading and error-checking mechanisms ensure near
perfect fidelity for DNA replication.
In a cell, DNA replication begins at specific locations, or origins of replication,
in the genome. Unwinding of DNA at the origin and synthesis of new strands,
accommodated by an enzyme known as helicase, results in replication
forks growing bi-directionally from the origin. A number of proteins are associated
with the replication fork to help in the initiation and continuation of DNA synthesis.
Most prominently, DNA polymerase synthesizes the new strands by adding
nucleotides that complement each (template) strand. DNA replication occurs during
the S-stage of interphase.
DNA replication (DNA amplification) can also be performed in
vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial
DNA primers can be used to start DNA synthesis at known sequences in a template
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DNA: Replication, DNA molecule. Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR),
Enzymology and Types
And Transcription-Mediated Amplification (TMA) are examples.
In this unit, you will study about replication of DNA- semiconservative mode,
Meselson - Stahl experiment. Enzymology of DNA replication - DNA polymerase
NOTES
I, II and III, topoisomerase I and II, helicase, primase, gyrase. Molecular basis of
DNA replication - replication fork, origin, Okazaki fragments. Types of replication
- circular and theta.

2.1 OBJECTIVES

After going through this unit, you will be able to:

• Discuss about replication of DNA
• Explain enzymology of DNA replication
• Understand the molecular basis of DNA replication

2.2 GENERAL INTRODUCTION

DeoxyriboNucleic Acid (DNA) is a molecule composed of two chains that coil

around each other to form a double helixcarrying the geneticinstructions used in
the growth, development, functioning, are reproduction of all known living
organisms and many viruses. DNA and RiboNucleic Acid (RNA) are nucleic
acids; alongside proteins, lipids and complex carbohydrates (polysaccharides),
nucleic acids are one of the four major types of macromoleculesthat are essential
for all known forms of life.
The two DNA strands are also known as polynucleotidesas they are
composed of simpler monomericunits called nucleotides. Each nucleotide is
composed of one of four nitrogen-containing nucleobases (Cytosine[C], Guanine[G],
Adenine[A] or Thymine[T]), a sugarcalled deoxyribose, and a phosphate group.
The nucleotides are joined to one another in a chain by covalent bondsbetween the
sugar of one nucleotide and the phosphate of the next, resulting in an alternating
sugar-phosphate backbone. The nitrogenous bases of the two separate
polynucleotide strands are bound together, according to base pairing rules (A with
T and C with G), with hydrogen bonds to make double-stranded DNA.
History
Nucleic acids were first isolated by Friedrich Miescher (1869) from pus cells.They
were named nuclein. Hertwig (1884) proposed nuclein to be the carrier of
hereditary traits. Because of their acidic nature they were named nucleinic acids
and then nucleic acids (Altmann, 1899).
Fisher (1880s) discovered the presence of purine and pyrimidine bases in
nucleic acids. Levene (1910) found deoxyribose nucleic acid to contain phosphoric
acid as well as deoxyribose sugar.
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 He characterised four types of nucleotides present in DNA. In 1950, DNA: Replication,
Enzymology and Types
Chargaff found that purine and pyrimidine content of DNA was equal. By this time
W.T. Astbury had found through X-ray diffraction that DNA is a polynucleotide
with nucleotides arranged perpendicular to the long axis of the molecule and
separated from one another by a distance of 0.34 nm. NOTES

In 1953, Wilkins and Franklin got very fine X-ray photographs of DNA.
The photographs showed that DNA was a helix with a width of 2.0 nm. One turn
of the helix was 3.4 nm with 10 layers of bases stacked in it. Watson and Crick
(1953) worked out the first correct double helix model from the X-ray photo­graphs
of Wilkins and Franklin. Wilkins, Watson and Crick were awarded Nobel Prize
for the same in 1962.
Watson and Crick (1953) built a 3D, molecular model of DNA that satisfied
all the details obtained from X-ray photographs. They proposed that DNA consisted
of a double helix with two chains having sugar phosphate on the outside and nitrogen
bases on the inner side.
The nitrogen bases of the two chains formed complementary pairs with
purine of one and pyrimidine of the other held together by hydrogen bonds (A-T,
C-G). Complementary base pairing between the two polynucleotide chains is
considered to be hall mark of their proposition. It is of course based on early
finding of Chargaff that A = T and Ñ = G Their second big proposal was that the
two chains are antiparallel with 5′ → 3′ orien­tation of one and Y → 5′orientation
of the other.
The two chains are twisted helically just as a rope ladder with rigid steps
twisted into a spiral. Each turn of the spiral contains 10 nucleotides. This double
helix or duplex model of DNA with antiparallel polynucleotide chains having
complementary bases has an implicit mechanism of its replication and copying.
Here both the polynucleotide chains function as templates forming two double
helices, each with one parent chain and one new but complementary strand. The
phenomenon is called semiconservative replication. In-vitro synthesis of DNA has
been carried out by Kornberg in 1959.
Types of DNA
DNA duplex model proposed by Watson and Crick is right handed spiral and is
called B-DNA (Balanced DNA). In the model the base pairs lie at nearly right
angles to the axis of helix. Another right handed duplex model is A-DNA (Alternate
DNA). Here, a single turn of helix has 11 base pairs.
The base pairs lie 20° away from perpendicular to the axis. C-DNA has 9
base pairs per turn of spiral while in D-DNA the number is only 8 base pairs. Both
are right handed. Z-DNA (Zigzag DNA) is left-handed double helix with zigzag
back-bone, alternate purine and pyrimidine bases, single turn of 45 Å length with
12 base pairs and a single groove (Refer Figure 2.1).
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DNA: Replication,
Enzymology and Types B-DNA is more hydrated and most frequently found DNA in living cells. It
is physiologically and biologically active form. However, it can get changed into
other forms. Right handed DNA is known to change temporarily into the left handed
form at least for a short distance. Such changes may cause changes in gene
NOTES
expression.

Fig. 2.1 Orientation of Adjacent Sugar Molecules in B and Z DNA

Circular and Linear DNA

In many prokaryotes the two ends of a DNA duplex are covalently linked to form
circular DNA. Circular DNA is naked, that is, without association with histone
proteins, though polyamines do occur. In linear DNA the two ends are free. It is
found in eukaryotic nuclei where it is associated with histone proteins.
Linear DNA, without association with histone proteins, also occurs in some
prokaryotes, for example Mycoplasma. In semi-autonomous cell organelles
(mitochondria, plastids) DNA is circular, less commonly linear. It is always naked.
Functions of DNA
• Genetic Information (Genetic Blue Print): DNA is the genetic material
which carries all the hereditary information. The genetic information is coded
in the arrangement of its nitrogen bases.
• Replication: DNA has unique property of replication or production of
carbon copies (Autocatalytic function). This is essential for transfer of genetic
information from one cell to its daughters and from one generation to the
next.
• Chromosomes: DNA occurs inside chromosomes. This is essential for
equitable distribution of DNA during cell division.
• Recombinations: During meiosis, crossing over gives rise to new
combination of genes called recombinations.
• Mutations: Changes in sequence of nitrogen bases due to addition, deletion
or wrong replication give rise to mutations. Mutations are the fountain head
of all variations and evolution.
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 • Transcription: DNA gives rise to RNAs through the process of DNA: Replication,
Enzymology and Types
transcription. It is heterocatalytic activity of DNA.
• Cellular Metabolism: It controls the metabolic reactions of the cells through
the help of specific RNAs, synthesis of specific proteins, enzymes and
hormones. NOTES
• Differentiation: Due to differential functioning of some specific regions of
DNA or genes, different parts of the organisms get differentiated in shape,
size and functions.
• Development: DNA controls development of an organism through working
of an internal genetic clock with or without the help of extrinsic information.
• DNA Finger Printing: Hypervariable microsatellite DNA sequences of
each individual are distinct. They are used in identification of individuals and
deciphering their relationships. The mechanism is called DNA finger printing.
• Gene Therapy: Defective heredity can be rectified by incorporating correct
genes in place of defective ones.
• Antisense Therapy: Excess availability of anti-mRNA or antisense RNAs
will not allow the pathogenic genes to express themselves. By this technique
failure of angioplasty has been checked. A modification of this technique is
RNA interference (RNAi).

Check Your Progress

1. What is DNA?
2. What did Fisher discover?
3. Give the contribution of Watson and Crick.

2.3 DNA REPLICATION: AN INSIGHT

The basis of inheritance of hereditary characters is DNA replication. It is a process

in which DNA makes its own exact copies via an autocatalytic process. The concept
of autocatalytic function is fulfilled when DNA makes two daughter molecules of
itself. This process is known to take place during the S- phase of cell cycle. Before
cell division, DNA so as to provide same compliment of DNA to both daughter
cells and they form the same set of genes just like the parental cell. Replication of
DNA helps in duplicating the entire genome once during every cell cycle (Refer
Figure 2.2). This is why, each chromosome has a pair of chromatids connected via
a centromere. These chromatids separate equally during the anaphase stage of
meiosis II. The mechanism of this replication process can be inferred from the
Double Helix Structure of DNA given by Watson and Crick. It says that the
DNA has two chains which are connected via hydrogen bonds. During replication,
these bonds are broken separating the two chains. The purines get separated from
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DNA: Replication, their respective pyrimidine counterparts but the sugar phosphate backbone does
Enzymology and Types
not break. The two separated chains act as templates and help in the formation of
two similar daughter DNA helices.

NOTES

Fig. 2.2 A Simplified Diagram Showing Replication of DNA

Modes of DNA Replication

There are three known modes of DNA replication. They are as follows:
• Semiconservative Mode
• Conservative Mode
• Dispersive Mode
Semiconservative Mode
This mode of DNA replication was given by Watson and Crick (1953) along
with their famous double helix structure of DNA.
This process starts with breaking of hydrogen bonds, present between the
two chains of DNA helix, from one end to the other. Each chain combines with the
complimentary chain (derived from nuclear sap) and leads to the formation of
daughter DNA helix.
Hence, each daughter helix contains one parental strand and one new
complimentary strand. This means that the parental DNA is semi conserved in the
daughter DNA.
Evidences showing semiconservative mode of replication:
Meselson-Stahl Experiment
The evidence for semiconservative replication was first shown by Mathew Meselson
and Franklin Stahl in 1958.
They grew cells of Escherichia coli on a medium containing heavy isotope
of nitrogen, N-15 in the form of Ammonium chloride. They allowed these cells to
grow for 14 generations so that this heavy isotope is incorporated in the DNA.
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 The DNA containing this isotope has higher density (1.724 gm/cm2) than DNA: Replication,
Enzymology and Types
the DNA containing normal N-14 (1.710 gm/cm2).
Hence, these are called heavy and light DNA respectively. These DNA
were subjected to equilibrium density gradient centrifugation and the centrifuge
NOTES
tubes contained distinct bands of the two DNAs.
These cells were then grown in medium containing normal N-14 and DNA
was extracted from respective dividing cell generations. The DNAs extracted from
each cell generation (1, 2, 3...) were analysed in CsCl density gradient.
The DNA that contained N-15 in both strands (heavy DNA) makes a band
that is present in high density position in the CsCl density gradient. On the other
hand, the one with light DNA makes a band at low density position. The DNA with
one N-15 strand and one N-14 strand (hybrid DNA) forms bands at intermediate
density.
When allowed to grow in N-14 medium for two cell generations, half of the
DNA bands were seen at hybrid/ intermediate density and other half were seen at
light density.
Hence, this experiment clearly provides evidence for semiconservative nature
of DNA replication because in case of conservative mode no intermediate bands
would have formed while in case of dispersive mode no light or intermediate bands
would have been formed (Refer Figure 2.3).

Fig. 2.3 Meselson and Stahl Experiment showing

Semiconservative Mode of DNA Replication
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DNA: Replication, Conservative Mode of DNA Replication
Enzymology and Types
This mode of replication states that the process is conserved and that the parental
DNA is completely conserved and it regulates the synthesis of daughter DNA helix.
NOTES Dispersive Mode of DNA Replication
This mode states that the parental DNA breaks into several pieces and each piece
replicates with segments to form daughter helices. Thus, the daughter DNA contains
both parental and new pieces of DNA.
Figure 2.4 illustrate the difference between three modes of replication.

Fig. 2.4 Figure Depicting Difference Between three Modes of Replication, i.e.,
Conservative, Semiconservative and Dispersive Mode of Replication

Check Your Progress

4. What is the basis of inheritance of hereditary?
5. What is replication?
6. How semiconservative process starts?

2.4 ENZYMOLOGY OF DNA REPLICATION

Enzymes are known to catalyze more than 5,000 biochemical reaction types.
Most enzymes are proteins, although a few are catalytic RNA molecules. The
latter are called ribozymes. Enzymes specificity comes from their unique three-
dimensional structures.
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 The various enzymes used in molecular biology are DNA polymerase, DNA: Replication,
Enzymology and Types
helicase, primase, ligase, exonuclease and endonuclease which are explained below:
DNA Polymerases
DNA polymerase is an enzyme that synthesizes DNA molecules from NOTES
deoxyribonucleotides, the building blocks of DNA. DNA molecules are the troves
of genetic information of an organism. DNA is the basis of life and is transferred
from parent to offspring’s. The DNA content of the parent is doubled by means of
replication mechanism aided by a specific enzyme, DNA polymerases. DNA
polymerase plays a central role in process of life and carries a weighty responsibility
of making an accurate copy of the cell’s genome. The DNA polymerases are
enzymes molecules by assembling nucleotides, the building blocks of DNA. The
first evidence of the existence of an enzymatic activity capable of synthesizing
DNA came in 1958 with the discovery of E. coli Pol I by A. Kornberg and
colleagues. DNA polymerase moves along the old strand in the 3'-5' direction,
creating a new strand having a 5'-3' direction.

Fig. 2.5 DNA Replication

Types and Roles of DNA Polymerases (Replicative and Repairative)

1. Prokaryotic DNA Polymerase
Prokaryotes contain five different types of DNA polymerase. These are described
below:
Pol I
• Polymerase I is a DNA repair enzyme from the family A polymerases that
has a 5' to 3' and 3' to 5' activity.
• Pol I accounts for more than 95% of polymerase activity in coli, although
cells that lack this polymerase have been found and its activity can be replaced
by the other four types of polymerase.
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DNA: Replication, • This DNA polymerase has a poor processivity rate, adding around 15 to
Enzymology and Types
20 nucleotides per second.
• This repair polymerase is involved in excision repair with 3'-5' and 5'-3'
exonuclease activity and processing of Okazaki fragments.
NOTES
Pol II
• DNA polymerase II, a Family B polymerase. Polymerase II is a DNA
repair enzyme with a 3' to 5' exonuclease activity.
• When DNA acquires damage in the form of short gaps, which block Pol III
activity, Pol II helps to remedy this problem by restarting DNA synthesis
downstream of these gaps.
• Pol II has 3'-5' exonuclease activity and participates in DNA repair,
replication restart to bypass lesions, and its cell presence can jump from
~30-50 copies per cell to ~200-300 during SOS induction.
Pol III
• This holoenzyme is the main polymerase in coli DNA replication and is one
of the family C polymerases.
• Polymerase III is the exonucleolytic proofreader, and can process of both
the leading and lagging DNA strands.
Pol IV
• This enzyme belongs to the Y family of DNA polymerases.
• Pol IV is an error-prone polymerase that has no 3' to 5' proofreading activity
and is involved in mutagenesis or the altering of DNA to give rise to a
mutation.
Pol V
• Pol V also belongs to the Y family of polymerases and allows DNA damage
to be bypassed in order for replication to continue.
• It is involved in SOS response and translation synthesis DNA repair
mechanisms.
2. Eukaryotic DNA Polymerase

POL α
• POL α is a members of Family B Polymerases and are the main polymerases
involved with nuclear DNA replication.
• This unique enzyme has two distinct polymerase activities: a 5'- 3' DNA-
dependent DNA polymerase, and a 5'- 3'.
• DNA-dependent RNA polymerase. The RNA polymerase activity is a
primase. Because of this, the enzyme is often referred to as Pol a: primase.
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 It is the only enzyme known to have both DNA polymerase and primase DNA: Replication,
Enzymology and Types
activities, and the only one capable of selfprimed DNA synthesis on a
previously unprimed ssDNA.
• Pol α does not have an intrinsic 3'- 5' exonuclease activity and also lacks a
NOTES
5'- 3' exonuclease activity. In-vivo, the primary function of Pol α: primase
is to make short RNA/DNA primers for replicative DNA synthesis.
DNA Polymerase β
• Belongs to family X polymerases are found mainly in vertebrates, and a few
are found in plants and fungi.
• Pol β is required for short-patch base excision repair, a DNA repair pathway
that is essential for repairing alkylated or oxidized bases as well as a basic
sites.
• This is the smallest and simplest of the classical eukaryotic polymerases; it
is composed of a single ~40-48 kDa protein.
• Pol β is not highly active and is not very processive. It has no intrinsic
exonuclease activities.
• Its preferred template is duplex DNA with short gaps, although it can bind
a nicked duplex and is capable of some limited displacement synthesis. Pol
β is primarily involved in DNA repair.
Polymerases λ, σ and µ (Lambda, Sigma, and Mu)
• Family X polymerases also contain the well-known eukaryotic
polymerasesuch as Pol σ (sigma), Pol λ (lambda)
• Pol σ and Pol µ, are involved in non-homologous end-joining, a mechanism
for rejoining DNA double-strand breaks due to hydrogen peroxide and
ionizing radiation, respectively.

Polymerases η, ι and κ (eta, iota, and kappa)

• Pol η (eta), Pol ι (iota), and Pol κ (kappa), are Family Y DNA polymerases
involved in the DNA repair by translation synthesis.
• Polymerases in Family Y are low-fidelity polymerases, but have been proven
to do more good than harm as mutations that affect the polymerase can
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DNA: Replication, cause various diseases, such a skin cancer and Xeroderma Pigmentosum
Enzymology and Types
Variant (XPS).
• Pol η is particularly important for allowing accurate translesion synthesis of
DNA damage resulting from ultraviolet radiation
NOTES
• Pol κ is thought to act as an extender or an inserter of a specific base at
certain DNA lesions.
Polymerases ζ (zeta)
• Pol ζ another B family polymerase, is involved in translesion synthesis.
• Pol ζ lacks 3' to 5' exonuclease activity, is unique in that it can extend
primers with terminal mismatches.
Polymerases γ and θ (gamma and theta)
• Pol γ (gamma) and Pol θ (theta) are Family A polymerases.
• Pol γ, is the only mtDNA, polymerase and therefore replicates, repairs,
and has proofreading 3'-5' exonuclease.
• Any mutation that leads to limited or non-functioning Pol γ has a significant
effect on mtDNA and is the most common cause of autosomal inherited
mitochondrial disorders.
• Pol θ, found in eukaryotes, its function is not clearly understood. Pol θ
belongs to Family A polymerase.
• Pol θ extends mismatched primer termini and can bypass abasic sites by
adding a nucleotide.
3. Reverse Transcriptase (RT)

Retrovirus Reverse Transcriptase

• Retroviruses package their genomes as ssRNA but replicate this RNA
through a dsDNA intermediate. To do this, they employ an RNA dependent
DNA polymerase (reverse transcriptase).
• RT is a typical DNA polymerase in the 5'- 3' direction of synthesis,
requirement for a template primer with a 3'-OH terminus, and requirements
for dNTPs and Mg2+.
• RT does not have a detectable DNA specific exonuclease activity, and
therefore has no proofreading function. As a result, its error rate is relatively
high. This is reflected in a high mutation rate; virus variants are generated at
high frequency. This is an important aspect of pathogenesis: retroviruses are
adept at evading host immunosurveillance because of high mutation frequency.
RT is unique among DNA polymerases in at least two respects:
o It can use primer, natural ssRNAs as template. It can also use a primed
ssDNA as template.
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 o It has intrinsic RNase H activity. RNase H is a processive exonuclease DNA: Replication,
Enzymology and Types
that specifically degrades the RNA strand of a DNA-RNA hybrid
beginning from either the 5' or 3' end
o It can also act as an endonuclease. RNase H hydrolyzes phosphodiester
NOTES
bonds to leave products with 3' hydroxyl and 5' phosphate ends.
o The enzyme is relatively processive and can replicate the 8 kb retrovirus
genome without a processivity factor.
Telomerase
Telomerase is a ribonucleoprotein recruited to replicate ends of linear chromosomes
because normal DNA polymerase cannot replicate the ends, or telomere.
Telomerase acts like other DNA polymerases by extending the 3' end, but, unlike
other DNA polymerases, telomerase does not require a template.
The protein and RNA components make up an active enzyme of ~200
kDa. The RNA component (~1.3 kb in yeast) contains the template sequence that
is used for DNA synthesis. (So telomerase carries its own template.) In vitro,
telomerase from a given species synthesizes the G-rich strand sequence
characteristic of the species. However, telomerase activity is present in actively
dividing cells, including immortalized (transformed) cells in culture, and in most
cancer cells. So telomerase may be useful for cancer diagnostics, and is a possible
target for therapeutics.
Significance of DNA Polymerases in Molecular Biology
DNA polymerases also play central roles in modern molecular biology and
biotechnology, enabling techniques including DNA cloning, the Polymerase Chain
Reaction (PCR), DNA sequencing, Single Nucleotide Polymorphism (SNP)
detection, Whole Genome Amplification (WGA), synthetic biology, and molecular
diagnostics.
Thermostable DNA Polymerase
Taq DNA polymerase. The original report of this enzyme, purified from the hot
springs bacterium Thermus aquaticus, was published in 1976. Later, the
polymerase chain reaction was developed and shortly thereafter ‘Taq’ became a
household word in molecular biology circles.
The thermophilic DNA polymerases, like other DNA polymerases, catalyze
template-directed synthesis of DNA from nucleotide triphosphates. A primer having
a free 3' hydroxyl is required to initiate synthesis and magnesium ion is necessary.
In general, they have maximal catalytic activity at 75 to 80°C, and substantially
reduced activites at lower temperatures. At 37°C, Taq polymerase has only about
10% of its maximal activity. In addition to Taq DNA polymerase, several other
thermostable DNA polymerases have been isolated and expressed from cloned
genes. Three of the most-used polymerases are described in the following
Table 2.2
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DNA: Replication, Table 2.2 Most Used Polymerases
Enzymology and Types

NOTES

DNA Helicases
In all cellular organisms from bacteria to humans, genetic information is locked
within a double helix formed by the two antiparallel DeoxyriboNucleic Acid (DNA)
strands. Although double-stranded DNA (dsDNA) is the form most suitable for
secure information storage, hydrogen bonds formed between complementary bases
(Watson-Crick base pairing) impair readout of this information by the cellular
machinery, which frequently requires a single-stranded DNA (ssDNA) intermediate
as a template. The unwinding of dsDNA into ssDNA, a function critical for virtually
every aspect of cellular DNA metabolism from RNA synthesis to homologous
DNA recombination, is provided by a ubiquitous class of enzymes called DNA
helicases. First identified in the 1970s, DNA helicases are motor proteins (often
called DNA motors) that convert chemical energy into mechanical work. Chemical
energy is derived from the hydrolysis of Adenosine TriphosPhate (ATP) or other
nucleoside triphosphates, and is coupled with mechanical work during at least
two important steps within the helicase reaction cycle:
• The unidirectional translocations along the substrate molecule.
• The melting of the DNA duplex, which together result in the formation of
the ssDNA intermediates essential for vital cellular processes (Refer Figure
2.6):

Fig. 2.6 Schematic Representation of the Helicase Reaction

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 The above figure describes about the helicase enzyme translocates along DNA: Replication,
Enzymology and Types
the DNA molecule and separates the strands. Energy for this unfavorable reaction
is provided by the hydrolysis of Adenosine TriphosPhates (ATP) to Adenosine
DiPhosphates (ADP) and inorganic Phosphate ions (Pi). In the presence of a
single-stranded DNA binding protein, reannealing of the DNA duplex is prevented. NOTES
The helicase depicted here displays a 3 → 5 polarity, tracking unidirectionally
along the lower of the two DNA strands in the duplex.
Classifications
Helicases are divided into five main superfamilies based on the presence and
composition of conserved amino acid (helicase signature) motifs. (It is important
to note, however, that only a small fraction of these putative helicases have been
studied biochemically and, of those proteins, not all have been shown to possess
nucleic acid strand separation activity). Biochemical and structural data have
suggested that helicases function as monomers, dimers, and multimers
(predominantly hexamers) and that they can also be classified based on a substrate
requirement for dsDNA, dsRNA, or DNA-RNA hybrids. To unwind dsDNA
efficiently, many DNA helicases need to initiate from an ssDNA region adjacent to
the duplex part of the substrate molecule. Based on the requirement for an ssDNA
overhang of a certain polarity, helicases are further divided into two functional
groups: those that utilize a 3 - terminated ssDNA are designated as 3→5 helicases,
whereas enzymes that require a 5 overhang are designated as 5→3 helicases.
Directional Translocation
It is now generally believed that the observed polarity requirement of helicases is
a consequence of a directional bias in translocation on ssDNA. For example, the
enzyme depicted is a 3→5 helicase. Upon binding to the ssDNA, it starts moving
toward the 5 end of the loading strand, which brings the enzyme to the
ssDNAdsDNA junction and subsequently through the duplex portion of the substrate
Evidence for directional translocation on ssDNA was provided by two different
approaches. The first examined the dependence of helicase ATPase activity on the
length of the ssDNA substrate; the second, based on the ability of many helicases
to create sufficient force during ssDNA translocation to disrupt the tight interaction
between streptavidin and biotin (Kd = 10–15M), measured the ability of the
helicase to increase the rate of streptavidin dissociation from DNA substrates
biotinylated at either the 3 or 5 end. This second method was used successfully to
determine the directionality of movement of several helicases on ssDNA. High-
resolution structural data suggest that the helicase signature motifs are not essential
for the duplex DNA separation per se, but for the ATP-dependent unidirectional
motion of the helicases on either single- or doublestranded DNA lattices.
Consequently, it has been proposed that the helicase signature motifs define a
modular structure that functions as the DNA motor, while additional domains,
which may vary from one protein to another, might be responsible for the DNA
unwinding Accessory factors. Once dsDNA unwinding is achieved, spontaneous
reannealing of the duplex may be avoided if the nascent ssDNA strands are trapped
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DNA: Replication, by single-stranded DNA binding proteins that hand off the intermediates to the
Enzymology and Types
next step in a reaction pathway. Although ssDNA binding proteins have frequently
been shown to stimulate helicase activity in vitro, helicase activity can also be
stimulated by other accessory factors that increase the rate or processivity of
NOTES unwinding. The primary replicative helicase of Escherchia coli, DNAB, is a good
example of a helicase that acts poorly in isolation from the accessory factors with
which the enzyme is intended to operate. As part of the replisome (the DNA
synthesis machinery of the cell), the role of DNAB is to separate the DNA strands
at the replication fork.
Primase
Primase is an enzyme that synthesizes short RNA sequences called primers. These
primers serve as a starting point for DNA synthesis. Since primase produces RNA
molecules, the enzyme is a type of RNA polymerase. Primase functions by
synthesizing short RNA sequences that are complementary to a single-stranded
piece of DNA, which serves as its template. It is critical that primers are synthesized
by primase before DNA replication can occur. This is because the enzymes that
synthesize DNA, which are called DNA polymerases, can only attach new DNA
nucleotides to an existing strand of nucleotides. Therefore, primase serves to prime
and lay a foundation for DNA synthesis. The enzyme is active only in the presence
of other proteins (including a helicase), which create a complex called the
primosome
DNA Ligase
Ends of DNA strands may be joined by the enzyme polynucleotide ligase, called
‘glue’ of the recombinant DNA molecule. The enzyme catalyses the formation of
a phosphodiester bond between the 3’OH and 5’P terminals of two nucleotides.
The enzyme is thus able to join unrelated DNA, repair nicks in single strand of
DNA and join the sugar phosphate backbones of the newly repaired and resident
region of a DNA strand.
The enzyme which is extensively used for covalently joining restriction
fragments is the ligase from Escherchia coli and that encoded by T4 phage. As
the main source of DNA ligase is T4 phage, hence, the enzyme is known as T4
DNA ligase.
The ligation reaction is controlled by several factors, such as pH, temperature,
concentration and kinds of sticky ends, etc. As ligase uses the ends of DNA
molecules as substrates rather than the entire DNA, the kinetics of joining depend
on the number of ends (concentration) available for joining.
Topoisomerases
Topoisomerases are enzymes that participate in the overwinding or underwinding
of DNA. The winding problem of DNA arises due to the intertwined nature of its
double-helical structure. During DNA replication and transcription, DNA becomes
overwound ahead of a replication fork. If left unabated, this torsion would eventually
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stop the ability of DNA or RNA polymerases involved in these processes to continue DNA: Replication,
Enzymology and Types
down the DNA strand.
In order to prevent and correct these types of topological problems caused
by the double helix, topoisomerases bind to DNA and cut the phosphate backbone
NOTES
of either one or both the DNA strands. This intermediate break allows the DNA
to be untangled or unwound, and, at the end of these processes, the DNA backbone
is resealed again. Since the overall chemical composition and connectivity of the
DNA do not change, the DNA substrate and product are chemical isomers, differing
only in their global topology, resulting in the name for these enzymes.
Topoisomerases are isomerase enzymes that act on the topology of DNA.
Bacterial topoisomerases and human topoisomerases proceed via similar
mechanisms for managing DNA supercoils.
Type I Topoisomerase
In molecular biology Type I topoisomerases are enzymes that cut one of the two
strands of double-stranded DNA, relax the strand, and reanneal the strand. They
are further subdivided into two structurally and mechanistically distinct
topoisomerases: type IA and type IB.
Type IA topoisomerases change the linking number of a circular DNA strand
by units of strictly 1.
Type IB topoisomerases change the linking number by multiples of 1 (n).
Historically, type IA topoisomerases are referred to as prokaryotic topo I,
while type IB topoisomerases are referred to as eukaryotic topoisomerase. This
distinction, however, no longer applies as type IA and type IB topoisomerases
exist in all domains of life.
Functionally, these subclasses perform very specialized functions. Prokaryotic
topoisomerase I (topo IA) can only relax negative supercoiled DNA, whereas
eukaryotic topoisomerase I (topo IB) can introduce positive supercoils, separating
the DNA of daughter chromosomes after DNA replication, and relax DNA.
Type II Topoisomerase
Type II topoisomerases cut both strands of the DNA helix simultaneously in order
to manage DNA tangles and supercoils. They use the hydrolysis of ATP,
unlike Type I topoisomerase. In this process, these enzymes change the linking
number of circular DNA by ±2.
Once cut, the ends of the DNA are separated, and a second DNA duplex
is passed through the break. Following passage, the cut DNA is religated. This
reaction allows type II topoisomerases to increase or decrease the linking number
of a DNA loop by 2 units, and it promotes chromosome disentanglement. Reactions
involving the increase in supercoiling require two molecules of ATP. For
example, DNA gyrase, a type II topoisomerase observed in Escherchia coli and
most other prokaryotes, introduces negative supercoils and decreases the linking
number by 2. Gyrase is also able to remove knots from the bacterial chromosome. Self-Instructional
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DNA: Replication, Along with gyrase, most prokaryotes also contain a second type IIA topoisomerase,
Enzymology and Types
termed topoisomerase IV. Gyrase and topoisomerase IV differ by their C-terminal
domains, which is believed to dictate substrate specificity and functionality for
these two enzymes. Footprinting indicates that gyrase, which forms a 140-base-
NOTES pair footprint and wraps DNA, introduces negative supercoils, while topoisomerase
IV, which forms a 28-base-pair footprint, does not wrap DNA.
Eukaryotic type II topoisomerase cannot introduce supercoils; it can only
relax them.
The roles of type IIB topoisomerases are less understood. Unlike type IIA
topoisomerases, type IIB topoisomerases cannot simplify DNA topology, but they
share several structural features with type IIA topoisomerases.
DNA Gyrase
DNA gyrase, or simply gyrase, is an enzyme within the class of topoisomerase
and is a subclass of Type II topoisomerases that reduces topological strain in an
ATP dependent manner while double-stranded DNA is being unwound by
elongating RNA-polymerase or by helicase in front of the progressing replication
fork. The enzyme causes negative supercoiling of the DNA or relaxes positive
supercoils. It does so by looping the template so as to form a crossing, then
cutting one of the double helices and passing the other through it before releasing
the break, changing the linking number by two in each enzymatic step. This process
occurs in prokaryotes (in particular, in bacteria), whose single circular DNA is cut
by DNA gyrase and the two ends are then twisted around each other to form
supercoils. Gyrase has been found in the apicoplast of the malarial
parasite Plasmodium falciparum, a unicellular eukaryote and in chloroplasts of
several plants. Bacterial DNA gyrase is the target of many antibiotics,
including nalidixic acid, novobiocin, and ciprofloxacin.
The unique ability of gyrase to introduce negative supercoils into DNA at
the expense of ATP hydrolysis is what allows bacterial DNA to have free negative
supercoils. The ability of gyrase to relax positive supercoils comes into play
during DNA replication and prokaryotic transcription. The helical nature of the
DNA causes positive supercoils to accumulate ahead of a translocating enzyme, in
the case of DNA replication, a DNA polymerase. The ability of gyrase
(and topoisomerase IV) to relax positive supercoils allows superhelical tension
ahead of the polymerase to be released so that replication can continue.
Gyrase Structure
DNA gyrase is a tetrameric enzyme that consists of 2 GyrA (or A subunit) and 2
GyrB (or B subunit) subunits. Structurally the complex is formed by 3 pairs of
‘gates’, sequential opening and closing of which results into the direct transfer of
DNA segment and introduction of 2 negative supercoils. N-gates are formed by
ATPase domains of GyrB subunits. Binding of 2 ATP molecules leads to dimerization
and, therefore, closing of the gates. Hydrolysis, on the contrary, opens them. DNA
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cleavage and reunion is performed by a catalytic center located in DNA-gates DNA: Replication,
Enzymology and Types
build by all gyrase subunits. C-gates are formed by GyrA subunits.

Check Your Progress

NOTES
7. What is DNA polymerase?
8. Give the process of polymerase I.
9. What is primase?

2.5 DNA REPLICATION MECHANISM:

PROKARYOTIC AND EUKARYOTIC

Comparing and Contrasting DNA Replication in Prokaryotes and

Eukaryotes
Replication of DNA – deoxyribonucleic acid – happens before a cell divides to
ensure that both cells receive an exact copy of the parent’s genetic material. While
there are many similarities in how prokaryotic and eukaryotic cells replicate their
DNA, there are several distinctions between them, due to the different size and
complexity of the molecules, including the time it takes to complete the process.
Differences Between Eukaryotic and Prokaryotic Cells
Prokaryotic cells are quite simple in structure. They have no nucleus, no organelles
and a small amount of DNA in the form of a single, circular chromosome. Eukaryotic
cells on the other hand, have a nucleus, multiple organelles and more DNA arranged
in multiple, linear chromosomes.
Steps in DNA Replication
DNA replication begins at a specific spot on the DNA molecule called the origin
of replication. At the origin, enzymes unwind the double helix making its components
accessible for replication. Each strand of the helix then separates from the other,
exposing the now unpaired bases to serve as templates for new strands. A small
segment of RNA – RiboNucleic Acid – is added as a primer, then new nucleotide
bases that complement the unpaired bases can be assembled to form two daughter
strands next to each parent strand. This assembly is accomplished with enzymes
called DNA polymerases. When the process is complete, two DNA molecules
have been formed identical to each other and to the parent molecule.
Similarities Between Prokaryotic and Eukaryotic DNA Replication
The steps for DNA replication are generally the same for all prokaryotic and
eukaryotic organisms. Unwinding the DNA is accomplished by an enzyme named
DNA helicase. Manufacturing new DNA strands is orchestrated by enzymes called
polymerases.
Both types of organisms also follow a pattern called semiconservative
replication. In this pattern, the individual strands of DNA are manufactured in
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DNA: Replication, different directions, producing a leading and a lagging strand. Lagging strands are
Enzymology and Types
created by the production of small DNA fragments called Okazaki fragments
that are eventually joined together. Both types of organisms also begin new DNA
strands with a small primer of RNA.
NOTES
Differences Between Prokaryotic and Eukaryotic DNA Replication
Differences between prokaryotic and eukaryotic DNA replication are largely
related to contrasts in size and complexity of the DNA and cells of these organisms.
The average eukaryotic cell has 25 times more DNA than a prokaryotic cell.
In prokaryotic cells, there is only one point of origin, replication occurs in
two opposing directions at the same time, and takes place in the cell cytoplasm.
Eukaryotic cells on the other hand, have multiple points of origin, and use
unidirectional replication within the nucleus of the cell. Prokaryotic cells possess
one or two types of polymerases, whereas eukaryotes have four or more.
Replication also happens at a much faster rate in prokaryotic cells, than in
eukaryotes. Some bacteria take only 40 minutes, while animal cells such as humans
may take up to 400 hours. In addition, eukaryotes also have a distinct process for
replicating the telomeres at the ends of their chromosomes. With their circular
chromosomes, prokaryotes have no ends to synthesize. Lastly, the short replication
in prokaryotes occurs almost continuously, but eukaryotic cells only undergo DNA
replication during the S-phase of the cell cycle.
DNA replication is fundamental process occurring in all living organism to
copy their DNA. The process is called replication in sense that each strand of ds
DNA serve as template for reproduction of complementary strand.
General Feature of DNA Replication
• DNA replication is semiconservative.
• It is bidirectional process.
• It proceed from a specific point called origin.
• It proceed in 5'-3' direction.
• It occur with high degree of fidelity.
• It is a multi-enzymatic process.
DNA Replication Occurs by Three Steps
1. Initiation
• Initiation complex formation.
• Closed complex formation.
• Open complex formation.
2. Elongation
• Leading strand synthesis.
• Lagging strand synthesis.
3. Termination
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DNA Replication in Prokaryotes DNA: Replication,
Enzymology and Types
1. Initiation
DNA replication begins from origin. In Escherichia coli, replication origin is called
OriC which consists of 245 base pair and contains DNA sequences that are highly NOTES
conserved among bacterial replication origin. Two types of conserved sequences
are found at OriC, three repeats of 13 bp (GATRCTNTTNTTTT) and four/five
repeats of 9 bp (TTATCCACA) called 13 mer and 9 mer respectively.
About 20 molecules of DNA A proteins binds with 9 mer repeats along
with ATP which causes DNA to wraps around DNAA protein forming initial
complex. The DNA A protein and ATP trigger the opening of 13 mer repeats
froming open complex.
Two copies of DNAB proteins (helicase) binds to 13 mer repeats. This
binding is facilitated by another molecule called DNAC. The DNAB-DNAC
interaction causes DNAB ring to open which binds with each of the DNA strand.
The hydrolysis of bound ATP release DNA leaving the DNAB bound to the DNA
strand (Refer Figure 2.7).

Fig. 2.7 Initiation - DNA Replication in Prokaryotes

The binding of helicase is key step in replication initiation. DNAB migrates

along the single stranded DNA in 5'-3' direction causing unwinding of the DNA.
The activity of helicase causes the topological stress to the unwinded strand
forming supercoiled DNA. This stress is relieved by the DNA topoisomerase
(DNA gyrase) by negative supercoiling. Similarly, single stranded binding protein
binds to th separated strand and prevents reannaeling of separated strand and
stabilize the strand.
The DNA polymerase cannot initiate DNA replication. So, at first primase
synthesize 10±1 nucleotide (RNA in nature) along the 5'-3' direction. In case of
E.coli primer synthesized by primase starts with ppp-AG-nucleotide. Primer is
closely associated with DNAB helicase so that it is positioned to make RNA
primer as ssDNA of lagging strand.
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DNA: Replication, 2. Elongation
Enzymology and Types
i. Leading Strand Synthesis
• Leading strand synthesis is more a straight forward process which begins
NOTES with the synthesis of RNA primer by primase at replication origin.
• DNA polymerase III then adds the nucleotides at 3'end. The leading
strand synthesis then proceed continuously keeping pace with unwinding
of replication fork until it encounter the termination sequences.
ii. Lagging Strand Synthesis
• The lagging strand synthesized in short fragments called Okazaki
fragments. At first RNA primer is synthesized by primase and as in leading
strand DNA polymerase III binds to RNA primer and adds dNTPS.
• On this level the synthesis of each Okazaki fragments seems straight
forward but the reality is quite complex (Refer Figure 2.8).

Fig. 2.8 Elongation - Replication Fork

Mechanism of Lagging Strand Synthesis

• The complexicity lies in the co-ordination of leading and lagging strand
synthesis. Both the strand are synthesized by a single DNA polymerase III
dimer which accomplished the looping of template (Refer Figure 2.9).

Fig. 2.9 Mechanism of Lagging Strand Aynthesis

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 • DNA of lagging strand synthesizing Okazaki fragments.Helicase (DNAB) DNA: Replication,
Enzymology and Types
and primase (DNAG) constitute a functional unit within replication complex
called primosome.
• DNA pol III use one set of core sub unit (Core polymerase) to synthesize
NOTES
leading strand and other set of core sub unit to synthesize lagging strand.
• In elongation steps, helicasein front of primaseand pol III, unwind the DNA
at the replication fork and travel along lagging strand template along 5'-3'
direction.
• DNAG primase occasionally associated with DNAB helicase synthesizes
short RNA primer. A new B-sliding clamp is then positioned at the primer
by B-clamp loading complex of DNA pol III.
• When the Okazaki fragments synthesis is completed, the replication halted
and the core sub unit dissociates from their sliding clamps and associates
with new clamp. This initiates the synthesis of new Okazaki fragments.
• Both leading and lagging strand are synthesized co-ordinately and
simultaneously by a complex protein moving in 5'-3' direction. In this way
both leading and lagging strand can be replicated at same time by a complex
protein that move in same direction.
• Every so often the lagging strands must dissociates from the replicosome
and reposition itself so that replication can continue.
• Lagging strand synthesis is not completes until the RNA primer has been
removed and the gap between adjacent Okazaki fragments are sealed. The
RNA primer are removed by exonuclease activity (5'-3') of DNA pol-I and
replaced by DNA. The gap is then sealed by DNA ligase using NAD as
co-factor.
Termination
• Evantually the two replication fork of circular E. coli chromosome meet at
termination recognizing sequences (ter).
• The Ter sequence of 23 bp are arranged on the chromosome to create trap
that the replication fork can enter but cannot leave. Ter sequences function
as binding site for TUS protein (Refre Figure 2.10).

Fig. 2.10 Termination - DNA Replication in Prokaryotes

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DNA: Replication, • Ter-TUS complex can arrest the replication fork from only one direction.
Enzymology and Types
Ter-TUS complex encounter first with either of the replication fork and halt
it. The other opposing replication fork halted when it collide with the first
one. This seems the Ter-TUS sequences is not essential for termination but
NOTES
it may prevents over replication by one fork if other is delayed or halted by
a damage or some obstacle.
• When either of the fork encounter Ter-TUS complex, replication halted.
• Final few hundred bases of DNA between these large protein complexes
are replicated by not yet known mechanism forming two interlinked
(cataneted) chromosome.
• In Escherchia coli DNA Topoisomerase IV (Type II) cut the two strand of
one circular DNA and segrate each of the circular DNA and finally join the
strand. The DNA finally transfer to two daughter cell.
DNA Replication in Eukaryotes
DNA replication in eukaryotes occur only in S-phase of cell cycle. However pre-
initiation occur in G1 pahse. Due to sheer size of chromosome in eukaryotes,
chromosome chromosome contains multiple origin of replication. ARS
(Autonomously Replicating Sequence) in case of yeast is origin for replication.
Steps in DNA Replication
1. Initiation
• The first steps is the formation of pre-initiation replication complex (pre-
RC). It occurs in two stage. Ist stage requires, there is no CDK activities. It
occur in early G1 phase. It is known as licensing but licensed pre-RC cannot
initiate replication at G1 phase. 2nd stage is binding of ORC (Origin
Recognition Complex).
• The replication begins with binding of ORC to the origin. ORC is a hexamer
of related protein and remains bounded even after DNA replication occurs.
Furthermore ORC is analogue of prokaryotic DNAA protein.
• After binding of ORC to origin, cdc6/cdc18 and cdtl coordinate the loading
of MEM (Mini chromosome maintainance) to origin.
• MEM complex is thought to be major eukaryotic helicase.
• After binding of MEM complex to pre-RC, cdtl get displaced. Then DdK
phosphorylates MEM, which activates its helicase activity. Again DdK and
CdK recruit another protein called cdc45 which then recruit all the DNA
replicating protein such that the origin get fired and replication begins (Refer
Figure 2.11).
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 DNA: Replication,
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NOTES

Fig. 2.11 Initiation Phase - DNA Replication in Eukaryotes

2. Elongation
• DNA polymerase δ synthesizes and adds dNTPs at 3' end of RNA primer.
• The leading and lagging strands are synthesized in the similar fashion as in
prokaryotic DNA replication ( Refer Figure 2.12).

Fig. 2.12 Elongation Phase - DNA Replication in Eukaryotes

3. Termination
• At the end of DNA replication the RNA primer are replaced by DNA by
5'-3' exonuclease and polymerase activity of DNA polymerase ε.
• Exonuclease activity of DNA polymerase removes the RNA primer and
polymerase activity adds dNTPs at 3'-OH end preceding the primer.
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DNA: Replication, • In case of bacteria, with circular genome, the replacement of RNA primer
Enzymology and Types
with DNA is not a problem because there is always a preceding 3'-OH in a
circular DNA.
• But in eukaryotic organism with linear DNA, there is a problem. When
NOTES
RNA primer at 5' end of daughter strand is removed, there is not a preceding
3'-OH such that the DNA polymerase can use it to replace by DNA. So, at
5' end of each daughter strand there is a gap (missing DNA). This missing
DNA cause loss of information contain in that region. This gap must be
filled before next round of replication.
• For solving this end replication problem;studies have found that linear end
of DNA called telomere has G:C rich repeats. These sequence is known as
telomere sequence. These repeats of telomere sequence is different among
different organisms. Telomere in human cell consists of repeats of TTAGGG/
AATCCC. Each species has its own species specific telomere repeats.
These telomere sequence donot codes anything but it is essential to fill the
gap in daughter strand and maintain the integrity of DNA.
Telomere Replication: End Replication Problem in Eukaryotic DNA
• There is an enzyme found in eukaryotic cell called telomerase.
• Telomerase is a DNA polymerase (RNA dependent DNA polymerase)
which adds many copies of telomere sequence at 3'-OH end of template
strand. Like other DNA polymerase, terlomerase also adds
deoxyribonucleotide at 3'-OH end. Unlike other DNA polymerase,
telomerase adds DNA at 3'-OH end of parent strand not at the daughter
strand and also it synthesizes the same sequences over and over in absence
of template strand (Refer Figure 2.13).

Fig. 2.13 Telomere Replication - End Replication Problem in Eukaryotic DNA

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 • First telomerase binds to 3'-OH end of parent strand by hybridization DNA: Replication,
Enzymology and Types
between its AACCCCAAC RNA sequences and TTGGGG DNA
sequences (telomere sequences of Tetrahymena).
• The telomerase adds TTG at 3' end of parent strand. After adding TTG
NOTES
sequences, telomerase translocates along 5'-3' end of parent strand. Now
the telomerase adds GGGTTG to 3' end by using its CCCAAC sequence.
Again telomerase translocates and adds GGGTTA sequence. This process
is continued for many time. The parent strand become more longer than
daughter strand. Now RNA polymerase (PRIMASE) synthesize RNA
primer by copying the parent strand in 5'-3' direction using telomere sequence
as template.
• The DNA polymerase can now extend the primer in 5'-3' direction by adding
deoxyribonucleotide to 3' end.
• The primer is now removed and it won’t be replaced because it is an extra
sequence added by copying telomere sequence.
• Finally the integrity of daughter strand is maintained.
Okazaki Fragments
Okazaki fragments are short sequences of DNA nucleotides (approximately 150
to 200 base pairs long in eukaryotes) which are synthesized discontinuously and
later linked together by the enzyme DNA ligase to create the lagging strand during
DNA replication. They were discovered in the 1960s by the Japanese molecular
biologists Reiji and Tsuneko Okazaki, along with the help of some of their
colleagues.
DNA Replication/Duplication
For normal cell growth and division in organisms, an initial step called DNA
replication is required. This process is based on semiconservative copy of DNA in
the nucleus of a growing cell.
DNA replication then begins with distortion of double helix by topoisomerase,
followed by separation of two DNA strands by helicase. Separation of DNA
single strands leads to formation of replication fork, where the replication machinery
(protein and enzyme complex) will bind. An RNA polymerase is the constituent of
this complex that adds small primers to the beginning of DNA single strand to be
copied. Then another enzyme, DNA polymerase, recognizes the primers added
by RNA polymerase and starts copying the DNA strands. At the end of replication
there are two DNA molecules, each of the double strand containing one strand
originating from cell and one copied (hence the name semiconservative replication).
However, a major hindrance to DNA replication comes from the fact that
DNA polymerase enzyme can only perform its function of nucleic acid
polymerization (addition of nucleic acids to the growing strand) in 5′→ 3′ sense.
Since the two strands of DNA molecule are antiparallel and replication fork
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DNA: Replication, separates the two strands at the same site, strand in the 5′→ 3′ direction is easily
Enzymology and Types
replicated towards the opening of replication fork. For this reason, this chain is
called ‘leader chain’. The problem lies in the other strand, which is oriented in
3′→ 5′ direction, in which DNA polymerase cannot bind and perform its function
NOTES because the opening of replication fork occurs in the opposite direction to
replication.
Okazaki Fragments Formation
To work around this problem, cell makes copies the strand that is oriented 3′→ 6′
in a discontinuous manner. This chain is called a ‘delayed chain’.
In this process, several small fragments of the delayed chain are replicated
as replication fork advances (5′→ 3′) and further separates the double helix from
DNA molecule. The fragments resulting from this discontinuous replication are
called ‘Okazaki Fragments’. Thus, although backward chain is growing in the
3′→ 5′ direction, in fact Okazaki Fragments are being synthesized in the 3′→ 5′
direction.
After the primers are removed, nucleic acid gaps between Okazaki
Fragments are filled and a final enzyme, DNA ligase, binds fragments to form a
single, single strand of continuous DNA ( Refer Figure 2.14) .

Fig. 2.14 DNA replication and Consequent Formation of Okazaki Fragments

Okazaki Fragments were named after the scientist who discovered them in
1969, Reiji Okazaki. In bacteria such fragments have a size of 1000 to 2000
nucleic acids; on the other hand, in eukaryotes have a size smaller than 200 nucleic
acids.
Difference Between Prokaryotic DNA Replication and Eukaryotic
DNA Replication
Some of the major Differences between Prokaryotic DNA Replication and
Eukaryotic DNA Replication are as follows:
Prokaryotic DNA Replication
• It occurs inside the cytoplasm (Refer Figure 2.15).
• There is single origin of replication.
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• DNA polymerase III carries out both initiation and elongation. DNA: Replication,
Enzymology and Types
• DNA repair and gap filling are done by DNA polymerase I (Refer Figure
2.16) .
NOTES

Fig. 2.15 Prokaryotic DNA Replication

Fig. 2.16 Replication Fork - Replication

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DNA: Replication, • RNA primer is removed by DNA polymerase I.
Enzymology and Types
• Okazaki fragments are large, 1000-2000 nucleotides long.
• Replication is very rapid, some 2000 bp per second.
NOTES • DNA gyrase is needed.
Eukaryotic DNA Replication
• It occurs inside the nucleus.
• Origin of replications are numerous.
• Initiation is carried out by DNA polymerase α while elongation by DNA
polymerase δ and ε.
• The same are performed by DNA polymerase β.
• RNA primer is removed by DNA polymerase β.
• Okazaki fragments are short, 100-200 nucleotides long.
• Replication is slow, some 100 nucleotides per second.
• DNA gyrase is not needed (Refer Figure 2.17).

Fig. 2.17 Eukaryotic DNA Replication

2.5.1 Types of Replication

There are two types of DNA replication, i.e., as follows:
• Circular DNA Replication
• Theta DNA Replication
Rolling Circle DNA Replication
In DNA replication, the DNA polymerase cannot initiate the synthesis of a new
DNA strand and must rely on a priming device. In general, an RNA primer is
synthesized at or near a replication origin to start synthesis of the leading strand.
However, a DNA primer terminus can be generated by a nuclease-generated nick
at a specific place in some circular duplex DNA, and replication will then proceed
unidirectionally, as shown in Figure 2.18. This mode of replication is called rolling
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circle replication and is found for replication of The Replicative Form (RF) form of DNA: Replication,
Enzymology and Types
bacteriophage single-stranded genomes of Gram-Negative Bacteria and of the
multicopy plasmids of Gram-Positive Bacteria. Rolling circle replication is also
observed in the late stage of the replication of the lambda phage genome and in the
process of the conjugative transfer of bacterial plasmids. NOTES

Fig. 2.18 Scheme for Rolling Circle Replication

As a summary, a typical DNA rolling circle replication has five steps:

• Circular dsDNA will be ‘nicked’.
• The 3' end is elongated using ‘unnicked’ DNA as leading strand (template);
5' end is displaced.
• Displaced DNA is a lagging strand and is made double stranded via a series
of Okazaki fragments.
• Replication of both ‘unnicked’ and displaced ssDNA.
• Displaced DNA circularizes.
Theta Structure
A Theta structure is an intermediate structure formed during the replication of a
circular DNA molecule (prokaryote DNA). Two replication forks can proceed
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DNA: Replication, independently around the DNA ring and when viewed from above the structure
Enzymology and Types
resembles the Greek letter ‘theta’ (θ). Originally discovered by John Cairns, it led
to the understanding that (in this case) bidirectional DNA replication could take
place. Proof of the bidirectional nature came from providing replicating cells with
NOTES a pulse of tritiated thymidine, quenching rapidly and then autoradiographing. Results
showed that the radioactive thymidine was incorporated into both forks of the
theta structure, not just one, indicating synthesis at both forks in opposite directions
around the loop.
Process of DNA Replication (Theta Model)
• Initiation of replication occurs at a specific region called origin of
replication where the ds DNA denatures to form ss DNA and within which
replication commences.
• The locally denatured segment of DNA is called the replication bubble and
the 2 strands in this region using which new complimentary strands are
synthesized are called the template strands.
• As the DNA unwinds, a y-shaped structure is formed at either ends of the
replication bubble. It is known as the replication fork. In such cases,
bidirectional replication occurs.
• The fork is generated by a complex of 7 proteins called primasome that
includes – DNA G primase, DNA B helicase, DNA C helicase assistant,
DNA T, Primase A, B and C.
• In coli, the OriC region spans 245 bp and contains clusters of 3 copies of
13-mer and 4 copies of 9-mer sequences.
• To initiate replication, an initiator protein called DNAA ATP (encoded by
DNAA gene) binds to 9-mer sequences and denatures the region connecting
it to 13-mer sequences by breaking A-T bonds which are weaker as they
are held by 2 hydrogen bonds. This requires energy from ATP. This forms
the initial complex.
• DNA helicase (DNA B) is loaded onto the DNA strands by a helicase
loader (DNA C). DNA helicase untwists the DNA. This forms the pre-
priming complex.
• SSBP (Single-Stranded Binding Protein) binds the open strands of DNA
to prevent rewinding. Gyrase, a type of topoisomerase, releases the tension
generated by rapid unwinding of the DNA strands at 3000 rpm.
• The DNA helicase recruits primase enzyme that synthesizes a short segment
of 5-10 nucleotides called primer which allows elongation of DNA. This
happens because DNA polymerase III can only add nucleotides but cannot
initiate synthesis of a new strand.
• Elongation is carried out by replisome which is made up of DNA polymerase
III and the primasome complex. The DNA pol III enzyme tethers itself to
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 the ss DNA via its core enzyme. The core enzyme catalyzes the DNA DNA: Replication,
Enzymology and Types
synthesis by adding complementary nucleotides.
• DNA synthesis takes place in 5' to 3' direction towards the replication fork.
The strand that is being synthesized in this direction continuously is called
NOTES
the leading strand while the strand that is synthesized in the opposite direction
is called lagging strand.
• The leading strand requires just 1 primer whereas the lagging strand requires
many such primers. Since, leading strand is synthesized continuously and
simultaneously along with the discontinuous synthesis of lagging strand, the
entire process of DNA synthesis is a semi-discontinuous process.
• Fragments of lagging strand are known as Okazaki fragments. After the
strands have been completely synthesized, these Okazaki fragments are
joined together.
• DNA pol III is removed. DNA pol I removes the primers by its 5' to 3'
exonuclease activity exposing the template nucleotides. It then adds
complementary nucleotides by its 5' to 3' polymerase activity to the 3'-OH
end of the previous Okazaki fragment, thereby replacing the primers.
• The nicks that remain behind are joined by ligase by creating a
phosphodiester bond. This is known as nick translation.
• Any errors in base-pairing is removed by DNA polymerase III by its 3' to
5' exonuclease activity immediately before proceeding onto the next
nucleotide. This is called proof-reading.
• Termination of this process occurs when the replication forks reach the ter
sites. TUS proteins (Terminus Utilization Substance) bind to ter sites and
halt progression of forks. In coli, there are 10 replication termini (Ter sites)
each spanning 23 bp.
• Ter B and C terminate the clockwise fork while ter A, D and E terminate
anti-clockwise fork.
• In circular chromosomes, the daughter chromosomes remain interlocked
and are called catenanes. Topoisomerase II resolves this problem by
breaking some bonds in DNA molecules so as to separate the strands –
Decatenation.

Check Your Progress

10. Give a difference between Eukaryotic and Prokaryotic cell.
11. Give a differences between Prokaryotic and Eukaryotic DNA replication.
12. What are the three steps of DNA replication?
13. How does DNA replication in Eukaryotes occur?
14. How does replication occur in Prokaryotes?
15. How does replication occur in Eukaryotes?
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DNA: Replication,
Enzymology and Types 2.6 ANSWERS TO CHECK YOUR PROGRESS
QUESTIONS

NOTES 1. DeoxyriboNucleic Acid is a molecule composed of two chains that coil

around each other to form a double helix carrying the genetic instructions
used in the growth, development, functioning, are reproduction of all known
living organisms and many viruses. DNA and RiboNucleic Acid (RNA) are
nucleic acids; alongside proteins, lipids and complex carbohydrates
(polysaccharides), nucleic acids are one of the four major types of
macromolecules that are essential for all known forms of life.
2. Fisher (1880s) discovered the presence of purine and pyrimidine bases in
nucleic acids. Levene (1910) found deoxyribose nucleic acid to contain
phosphoric acid as well as deoxyribose sugar.
3. Watson and Crick (1953) built a 3D, molecular model of DNA that satisfied
all the details obtained from X-ray photographs. They proposed that DNA
consisted of a double helix with two chains having sugar phosphate on the
outside and nitrogen bases on the inner side.
4. The basis of inheritance of hereditary characters is DNA replication.
5. Replication is a process in which DNA makes its own exact copies via an
autocatalytic process.
6. Semiconservative mode starts with breaking of hydrogen bonds, present
between the two chains of DNA helix, from one end to the other. Each
chain combines with the complimentary chain and leads to the formation of
daughter DNA helix.
7. DNA polymerase is an enzyme that synthesizes DNA molecules from
deoxyribonucleotides, the building blocks of DNA. DNA molecules are
the troves of genetic information of an organism.
8. Polymerase I process is as follows:
• Polymerase I is a DNA repair enzyme from the family A polymerases
that has a 5' to 3' and 3' to 5' activity.
• Pol I accounts for more than 95% of polymerase activity in coli, although
cells that lack this polymerase have been found and its activity can be
replaced by the other four types of polymerase.
• This DNA polymerase has a poor processivity rate, adding around 15
to 20 nucleotides per second.
• This repair polymerase is involved in excision repair with 3'-5' and 5'-3'
exonuclease activity and processing of Okazaki fragments.
9. Primase is an enzyme that synthesizes short RNA sequences called primers.
These primers serve as a starting point for DNA synthesis.
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10. Prokaryotic cells are quite simple in structure. They have no nucleus, no DNA: Replication,
Enzymology and Types
organelles and a small amount of DNA in the form of a single, circular
chromosome. Eukaryotic cells on the other hand, have a nucleus, multiple
organelles and more DNA arranged in multiple, linear chromosomes.
NOTES
11. Differences between prokaryotic and eukaryotic DNA replication are largely
related to contrasts in size and complexity of the DNA and cells of these
organisms. The average eukaryotic cell has 25 times more DNA than a
prokaryotic cell.
In prokaryotic cells, there is only one point of origin, replication occurs in
two opposing directions at the same time, and takes place in the cell
cytoplasm. Eukaryotic cells on the other hand, have multiple points of origin,
and use unidirectional replication within the nucleus of the cell. Prokaryotic
cells possess one or two types of polymerases, whereas eukaryotes have
four or more.
12. DNA Replication occurs by three steps:
1. Initiation
• Initiation complex formation
• Closed complex formation
• Open complex formation
2. Elongation
• Leading strand synthesis
• Lagging strand synthesis
3. Termination
13. DNA replication in eukaryotes occur only in S-phase of cell cycle. However
pre-initiation occur in G1 pahse. Due to sheer size of chromosome in
eukaryotes, chromosome chromosome contains multiple origin of replication.
ARS (Autonomously Replicating Sequence) in case of yeast is origin for
replication.
14. Prokaryotic DNA Replication:
• It occurs inside the cytoplasm.
• There is single origin of replication.
• DNA polymerase III carries out both initiation and elongation.
• DNA repair and gap filling are done by DNA polymerase I.
• RNA primer is removed by DNA polymerase I.
• Okazaki fragments are large, 1000-2000 nucleotides long.
15. Eukaryotic DNA Replication:
• It occurs inside the nucleus.
• Origin of replications are numerous.
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DNA: Replication, • Initiation is carried out by DNA polymerase á while elongation by DNA
Enzymology and Types
polymerase δ and ε.
• The same are performed by DNA polymerase β.
NOTES • RNA primer is removed by DNA polymerase β.
• Okazaki fragments are short, 100-200 nucleotides long.
• Replication is slow, some 100 nucleotides per second.
• DNA gyrase is not needed.

2.7 SUMMARY

• DeoxyriboNucleic Acid (DNA) is a molecule composed of two chains that

coil around each other to form a double helix carrying the genetic instructions
used in the growth, development, functioning, are reproduction of all known
living organisms and many viruses.
• DNA and RiboNucleic Acid (RNA) are nucleic acids; alongside proteins,
lipids and complex carbohydrates (polysaccharides), nucleic acids are one
of the four major types of macromolecules that are essential for all known
forms of life.
• Nucleic acids were first isolated by Friedrich Miescher (1869) from pus
cells. They were named nuclein. Hertwig (1884) proposed nuclein to be
the carrier of hereditary traits. Because of their acidic nature they were
named nucleinic acids and then nucleic acids (Altmann, 1899).
• Fisher (1880s) discovered the presence of purine and pyrimidine bases in
nucleic acids. Levene (1910) found deoxyribose nucleic acid to contain
phosphoric acid as well as deoxyribose sugar.
• The basis of inheritance of hereditary characters is DNA replication. It is a
process in which DNA makes its own exact copies via an autocatalytic
process.
• The concept of autocatalytic function is fulfilled when DNA makes two
daughter molecules of itself. This process is known to take place during the
S- phase of cell cycle.
• Before cell division, DNA so as to provide same compliment of DNA to
both daughter cells and they form the same set of genes just like the parental
cell.
• Replication of DNA helps in duplicating the entire genome once during
every cell cycle. This is why, each chromosome has a pair of chromatids
connected via a centromere. These chromatids separate equally during the
anaphase stage of meiosis II.
• The mechanism of this replication process can be inferred from the Double
Helix Structure of DNA given by Watson and Crick. It says that the DNA
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 has two chains which are connected via hydrogen bonds. During replication, DNA: Replication,
Enzymology and Types
these bonds are broken separating the two chains.
• The purines get separated from their respective pyrimidine counterparts but
the sugar phosphate backbone does not break.
NOTES
• The two separated chains act as templates and help in the formation of two
similar daughter DNA helices.
• Replication of DNA – DeoxyriboNucleic Acid – happens before a cell
divides to ensure that both cells receive an exact copy of the parent’s genetic
material.
• While there are many similarities in how prokaryotic and eukaryotic cells
replicate their DNA, there are several distinctions between them, due to
the different size and complexity of the molecules, including the time it takes
to complete the process.
• Prokaryotic cells are quite simple in structure. They have no nucleus, no
organelles and a small amount of DNA in the form of a single, circular
chromosome. Eukaryotic cells on the other hand, have a nucleus, multiple
organelles and more DNA arranged in multiple, linear chromosomes.
• DNA replication begins at a specific spot on the DNA molecule called the
origin of replication.
• At the origin, enzymes unwind the double helix making its components
accessible for replication.
• Each strand of the helix then separates from the other, exposing the now
unpaired bases to serve as templates for new strands.
• A small segment of RNA – RiboNucleic Acid – is added as a primer, then
new nucleotide bases that complement the unpaired bases can be assembled
to form two daughter strands next to each parent strand. This assembly is
accomplished with enzymes called DNA polymerases. When the process
is complete, two DNA molecules have been formed identical to each other
and to the parent molecule.
• The steps for DNA replication are generally the same for all prokaryotic
and eukaryotic organisms. Unwinding the DNA is accomplished by an
enzyme named DNA helicase. Manufacturing new DNA strands is
orchestrated by enzymes called polymerases.
• Lagging strands are created by the production of small DNA fragments
called Okazaki fragments that are eventually joined together. Both types of
organisms also begin new DNA strands with a small primer of RNA.
• Differences between prokaryotic and eukaryotic DNA replication are largely
related to contrasts in size and complexity of the DNA and cells of these
organisms. The average eukaryotic cell has 25 times more DNA than a
prokaryotic cell.
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DNA: Replication, • In prokaryotic cells, there is only one point of origin, replication occurs in
Enzymology and Types
two opposing directions at the same time, and takes place in the cell
cytoplasm.
• Eukaryotic cells on the other hand, have multiple points of origin, and use
NOTES
unidirectional replication within the nucleus of the cell. Prokaryotic cells
possess one or two types of polymerases, whereas Eukaryotes have four
or more.
• Replication also happens at a much faster rate in prokaryotic cells, than in
eukaryotes. Some bacteria take only 40 minutes, while animal cells such as
humans may take up to 400 hours. In addition, eukaryotes also have a
distinct process for replicating the telomeres at the ends of their
chromosomes.
• DNA replication is fundamental process occurring in all living organism to
copy their DNA. The process is called replication in sense that each strand
of ds DNA serve as template for reproduction of complementary strand.
• In Escherchia coli, replication origin is called OriC which consists of 245
base pair and contains DNA sequences that are highly conserved among
bacterial replication origin.
• DNA replication in eukaryotes occur only in S-phase of cell cycle. However
pre-initiation occur in G1 pahse.
• Due to sheer size of chromosome in eukaryotes, chromosome chromosome
contains multiple origin of replication. ARS (Autonomously Replicating
Sequence) in case of yeast is origin for replication.
• Okazaki fragments are short sequences of DNA nucleotides (approximately
150 to 200 base pairs long in Eukaryotes) which are synthesized
discontinuously and later linked together by the enzyme DNA ligase to create
the lagging strand during DNA replication.
• For normal cell growth and division in organisms, an initial step called DNA
replication is required. This process is based on semiconservative copy of
DNA in the nucleus of a growing cell.
• DNA replication then begins with distortion of double helix by to
poisomerase, followed by separation of two DNA strands by helicase.
Separation of DNA single strands leads to formation of replication fork,
where the replication machinery (protein and enzyme complex) will bind.
• An RNA polymerase is the constituent of this complex that adds small
primers to the beginning of DNA single strand to be copied. Then another
enzyme, DNA polymerase, recognizes the primers added by RNA
polymerase and starts copying the DNA strands.
• At the end of replication there are two DNA molecules, each of the double
strand containing one strand originating from cell and one copied (hence
the name semiconservative replication).
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 DNA: Replication,
2.8 KEY WORDS Enzymology and Types

• DNA polymerase: DNA polymerase is an enzyme that synthesizes DNA

molecules from deoxyribonucleotides, the building blocks of DNA. NOTES
• Telomere: A telomere is a region of repetitive nucleotide sequences at
each end of a chromosome, which protects the end of the chromosome
from deterioration or from fusion with neighboring chromosomes.
• Replication fork: The replication fork is a very active area where
DNA replication takes place. It is created when DNA helicase unwinds
the double helix structure of the DNA.
• Exonucleases: Exonucleases are enzymes that work by cleaving
nucleotides one at a time from the end of a polynucleotide chain.
• Okazaki fragments: Okazaki fragments are short sequences of DNA
nucleotides which are synthesized discontinuously and later linked together
by the enzyme DNA ligase to create the lagging strand during DNA
replication.

2.9 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is DNA polymerase?
2. Write in short about DNA helicase.
3. What are the three steps of DNA replication? Explain.
4. What are the similarities between Prokaryotic and Eukaryotic DNA
replication ?
5. Give the differences between Prokaryotic and Eukaryotic DNA replication.
6. Write the general feature of DNA replication.
7. What are the phases of DNA replication?
8. Write about initiation process of DNA replication in Eukaryotes.
Long Answer Questions
1. Discuss about DNA and its history.
2. Explain DNA replication.
3. Elaborate a note on enzymology of DNA replication.
4. Give a detailed account on mechanism of Prokaryotic and Eukaryotic DNA
replication.
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DNA: Replication, 5. Explain the DNA replication process in Prokaryotes in detail discussing
Enzymology and Types
about its phases.
6. Discuss the mechanism of leading and lagging strands synthesis in DNA
replication.
NOTES
7. Give a elaborated note on DNA replication process in Eukaryotes explaining
about its phases in detail.
8. Explain about Okazaki fragments in detail.
9. Distinguish between between Prokaryotic DNA replication and Eukaryotic
DNA replication.

2.10 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 RNA: Structure and Types

UNIT 3 RNA: STRUCTURE AND

TYPES
NOTES
Structure
3.0 Introduction
3.1 Objectives
3.2 RNA: Structure, Function and Types
3.3 Answers to Check Your Progress Questions
3.4 Summary
3.5 Key Words
3.6 Self Assessment Questions and Exercises
3.7 Further Readings

3.0 INTRODUCTION

RiboNucleic Acid (RNA) is a polymeric molecule essential in various biological

roles in coding, decoding, regulation and expression of genes. RNA
and DNA are nucleic acids, and, along with lipids, proteins and carbohydrates,
constitute the four major macromolecules essential for all known forms of life. Like
DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it is more
often found in nature as a single-strand folded onto itself, rather than a paired
double-strand. Cellular organisms use messenger RNA (mRNA) to convey genetic
information (using the nitrogenous bases of Guanine, Uracil, Adenine, and Cytosine,
denoted by the letters G, U, A, and C) that directs synthesis of specific proteins.
Many viruses encode their genetic information using an RNA genome.
Some RNA molecules play an active role within cells by catalyzing biological
reactions, controlling gene expression, or sensing and communicating responses
to cellular signals. One of these active processes is protein synthesis, a universal
function in which RNA molecules direct the synthesis of proteins on ribosomes.
This process uses transfer RNA (tRNA) molecules to deliver amino acids to the
ribosome, where ribosomal RNA (rRNA) then links amino acids together to form
coded proteins.
Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1'
through 5'. A base is attached to the 1' position, in general, Adenine (A),
Cytosine (C), Guanine (G), or Uracil (U). Adenine and guanine are purines,
cytosine and uracil are pyrimidines. A phosphate group is attached to the 3' position
of one ribose and the 5' position of the next. The phosphate groups have a negative
charge each, making RNA a charged molecule (polyanion). The bases
form hydrogen bonds between cytosine and guanine, between adenine and uracil
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RNA: Structure and Types
and between guanine and uracil. However, other interactions are possible, such as
a group of adenine bases binding to each other in a bulge, or the GNRA tetraloop
that has a guanine–adenine base-pair.
NOTES In this unit, you will study about structure of RNA, types of RNA- tRNA,
mRNA and rRNA.

3.1 OBJECTIVES

After going through this unit, you will be able to:

• Discuss the structure of RNA
• Explain the types of RNA - tRNA, mRNA and rRNA

3.2 RNA: STRUCTURE, FUNCTION AND TYPES

RiboNucleic Acid (RNA) is a polymeric molecule essential in various biological

roles in coding, decoding, regulation and expression of genes.RNA and
DNA are nucleic acids, and, along with lipids, proteins and carbohydrates,
constitute the four major macromolecules essential for all known forms of life. Like
DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it is more
often found in nature as a single-strand folded onto itself, rather than a paired
double-strand. Cellular organisms use messenger RNA (mRNA) to convey genetic
information (using the nitrogenous bases of guanine, uracil, adenine, and cytosine,
denoted by the letters G, U, A, and C) that directs synthesis of specific proteins.
Many viruses encode their genetic information using an RNA genome.
With the discovery of the molecular structure of the DNA double helix in
1953, researchers turned to the structure of RiboNucleic Acid (RNA) as the next
critical puzzle to be solved on the road to understanding the molecular basis of life.
RiboNucleic Acid (RNA) is a type of molecule that consists of a long chain of
nucleotide units. Each nucleotide consists of a nitrogenous base, a ribose sugar,
and a phosphate. RNA is very similar to DNA, but differs in a few important
structural details: in the cell, RNA is usually single-stranded, while DNA is usually
double-stranded; RNA nucleotides contain ribose while DNA contains deoxyribose
(a type of ribose that lacks one oxygen atom); and RNA has the base uracil rather
than thymine that is present in DNA.
RNA is transcribed from DNA by enzymes called RNA polymerases and is
generally further processed by other enzymes. RNA is central to the synthesis of
proteins. Here, a type of RNA called messenger RNA carries information from
DNA to structures called ribosomes. These ribosomes are made from proteins
and ribosomal RNAs, which come together to form a molecular machine that can
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read messenger RNAs and translate the information they carry into proteins. There RNA: Structure and Types

are many RNAs with other roles – in particular regulating which genes are expressed,
but also as the genomes of most viruses. Ribose Nucleic Acids Most cellular RNA
is single stranded, although some viruses have double stranded RNA. The single
NOTES
RNA strand is folded upon itself, either entirely or in certain regions. In the folded
region a majority of the bases are complementary and are joined by hydrogen
bonds. This helps in the stability of the molecule. In the unfolded region the bases
have no complements. Because of this RNA does not have the purine, pyrimidine
equality that is found in DNA. RNA also differs from DNA in having ribose as the
sugar instead of deoxyribose. The common nitrogenous bases of RNA are adenine,
guanine, cytosine and uracil. Thus the pyrimidine uracil substitutes thymine of DNA.
In regions where purine pyrimidine pairing takes place, adenine pairs with uracil
and guanine with cytosine. In addition to the four bases mentioned above, RNA
also has some unusual bases.
Chemical Structure of RNA
An important structural feature of RNA that distinguishes it from DNA is the
presence of a hydroxyl group at the 2' position of the ribose sugar. The presence
of this functional group causes the helix to adopt the A-form geometry rather than
the B-form most commonly observed in DNA. This results in a very deep and
narrow major groove and a shallow and wide minor groove. A second consequence
of the presence of the 2'-hydroxyl group is that in conformationally flexible regions
of an RNA molecule (that is, not involved in formation of a double helix), it can
chemically attack the adjacent phosphodiester bond to cleave the backbone. Most
cellular RNA molecules are single stranded. They may form secondary structures
such as stem-loop and hairpin ( Refer Figure 3.1).

Fig. 3.1 Secondary Structure of RNA - Stem-Loop and Hairpin

There are more unusual bases in RNA than in DNA. All normal RNA chains either
start with adenine or guanine: Three types of cellular RNA have been distinguished:
• Messenger RNA (mRNA) or template RNA
• Ribosomal RNA (rRNA)
• Soluble RNA (sRNA) or transfer RNA (tRNA)
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RNA: Structure and Types Ribosomal and transfer RNA comprise about 98% of all RNA. All three forms of
RNA are made on a DNA template. Transfer RNA and messenger RNA are
synthesized on DNA templates of the chromosomes, while ribosomal RNA is
derived from nucleolar DNA. The three types of RNA are synthesized during
NOTES different stages in early development. Most of the RNA synthesized during cleavage
is mRNA. Synthesis of tRNA occurs at the end or cleavage, and rRNA synthesis
begins during gastrulation.
Ribosomal RNA – rRNA
Ribosomal RNA, as the name suggests, is found in the ribosomes. It comprises
about 80% of the total RNA of the cell. The base sequence of rRNA is
complementary to that of the region of DNA where it is synthesized. In eukaryotes
ribosomes are formed on the nucleolus. Ribosomal RNA is formed from only a
small section of the DNA molecule, and hence there is no definite base relationship
between rRNA and DNA as a whole. Ribosomal RNA consists of a single strand
twisted upon itself in some regions. It has helical regions connected by intervening
single strand regions. The helical regions may show presence or absence of positive
interaction. In the helical region most of the base pairs are complementary, and
are joined by hydrogen bonds. In the unfolded single strand regions the bases
have no complements.
Ribosomal RNA contains the four major RNA bases with a slight degree of
methylation, and shows differences in the relative proportions of the bases between
species. Its molecules appear to be single polynucleotide strands which are
unbranched and flexible. At low ionic strength rRNA behaves as a random coil,
but with increasing ionic strength the molecule shows helical regions produced by
base pairing between adenine and uracil and guanine and cytosine.
Hence rRNA does not show purine-pyrimidine equality. The rRNA strands
unfold upon heating and refold upon cooling. Ribosomal RNA is stable for at least
two generations. The ribosome consists of proteins and RNA. The 70S ribosome
of prokaryotes consists of a 30S subunit and a 50S subunit. The 30S subunit
contains 16S rRNA, while the 50S subunit contains 23S and 5S rRNA. The 80S
eukaryote ribosome consists of a 40S and a 60S subunit. In vertebrates the 40S
subunit contains 18S rRNA, while the 60S subunit contains 28- 29S, 5.8S and
5S rRNA. In plants and invertebrates the 40S subunit contains 16- 18S RNA,
while the 60S subunit contains 25S and 58 and 5.8S rRNA.
There are three types of ribosomal RNA on the basis of sedimentation and
molecular weight. Two of these classes are high molecular weight RNAs, while
the third is a low molecular weight RNA. The three classes are:
• High molecular weight rRNA with molecular weight of over a million, for
example 21s-29s RNA,
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 • High molecular weight rRNA with molecular weight below a million, for RNA: Structure and Types

example 12-8-188 rRNA,

• Low molecular weight rRNA, for example 58 rRNA.
NOTES

Fig. 3.2 RNA Structure

Messenger RNA - mRNA

Jacob and Monod (1961) proposed the name messenger RNA for the RNA
carrying information for protein synthesis from the DNA (genes) to the sites of
protein formation (ribosomes). It consists of only 3 to 5% of the total cellular
RNA.
Size of Messenger RNA - mRNA
The molecular weight of an average sized mRNA molecule is about 500,000, and
its sedimentation coefficient is 8S. It should be noted however, that mRNA varies
greatly in length and molecular weight. Since most proteins contain at least a hundred
amino acid residues, mRNA must have at least 100 × 3= 300 nucleotides on the
basis of the triplet code.
Stability of Messenger RNA - mRNA
The cell does not contain large quantities of mRNA. This is because mRNA, unlike
other RNAs is constantly undergoing breakdown. It is broken down to its constituent
ribonucleotides by ribonucleases.
Structure of Messenger RNA
mRNA Messenger RNA is always single stranded. It contains mostly the bases
Adenine, Guanine, Cytosine and Uracil. There are few unusual substituted bases.
Although there is a certain amount of random coiling in extracted mRNA, there is
no base pairing. In fact base pairing in the mRNA strand destroys its biological
activity. Since mRNA is transcribed on DNA (genes), its base sequence is
complementary to that of the segment of DNA on which it is transcribed. This has
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RNA: Structure and Types been demonstrated by hybridization experiments in which artificial RNA, DNA
double strands are produced. Hydrization takes place only if the DNA and RNA
strands are complementary.
Usually each gene transcribes its own mRNA. Therefore, there are
NOTES
approximately as many types of mRNA molecules as there are genes. There may
be 1,000 to 10.000 different species of mRNA in a cell. These mRNA types
differ only in the sequence of their bases and in length. When one gene (cistron)
codes for a single mRNA strand the mRNA is said to be monocistronic. In many
cases, however, several adjacent cistrons may transcribe an mRNA molecule,
which is then said to be polycistronic or polygenic.
The mRNA molecule has the following structural features:
• Cap: At the 5' end of the mRNA molecule in most eukaryote cells and
animal virus molecules is found a ‘cap’. This is blocked methylated structure,
m7Gpp Nmp Np or m7Gpp Nmp Nmp Np. where: N = any of the four
nucleotides and Nmp = 20 methyl ribose. The rate of protein synthesis
depends upon the presence of the cap. Without the cap mRNA molecules
bind very poorly to the ribosomes.
• Noncoding Region 1 (NC1): The cap is followed by a region of 10 to
100 nucleotides. This region is rich in A and U residues, and does not
translate protein.
• Initiation Codon: It is AUG in both prokaryotes and eukaryotes.
• Coding Region: It consists of about 1,500 nucleotides on the average and
translates protein It is made up of 73-93 nucleotides (Rich and RajBhandary,
1976). Each bacterial cell probably contains about a hundred or more
different types of tRNA. The function of tRNA is to carry amino acids to
mRNA during protein synthesis. Each amino acid is carried by a specific
tRNA. Since 20 amino acids are coded to form proteins, it follows that
there must be at least 20 types of tRNA.
It was formerly thought that only 20 tRNA molecular types exist, one for
each amino acid. It has, however, been shown that in several cases there
are at least two types of tRNA for each amino acid. Thus there are many
more tRNA molecules than amino acid types. These are probably coded
by one gene.
Transfer RNA is synthesized in the nucleus on a DNA template. Only
0.025% of DNA codes for tRNA. Synthesis of tRNA occurs near the end
of cleavage stages. Transfer RNA is an exception to other cellular RNAs in
that a part of its ribonucleotide sequence (-CCA) is added after it comes
off the DNA template. Like rRNA, tRNA is also formed from only a small
section of the DNA molecule. Therefore, it does not show any obvious
base relationships to DNA. The tRNA molecule consists of a single strand
looped about it self. The 3' end always terminates in a -C-C-A (Cytosine-
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 Cytosine-Adenine) sequence. The 5' end terminates in G (Guanine) or C RNA: Structure and Types

(Cytosine). Many of the bases are bonded to each other, but there are also
unpaired bases.
Transfer RNA - tRNA or Soluble RNA NOTES
sRNA after rRNA the second most common RNA in the cell is transfer RNA. It is
also called soluble RNA because it is too small to be precipitated by
ultracentrifugation at 100,000 g. It constitutes about 10-20% of the total RNA of
the cell. Transfer RNA is a relatively small RNA having a molecular weight of
about 25,000 to 30,000 and the sedimentation coefficient of mature eukaryote
tRNA is 3.8S.
Structure of Transfer RNA – tRNA
The nucleotide sequence (primary structure) of tRNA was first worked out by
Holley et al. (1965) for yeast alanine tRNA. Since then the sequence of about 75
different tRNAs, ranging from bacteria to mammals, has been established. The
different tRNAs are all minor variants of the same basic type of structure. Several
models of the secondary structure of tRNA have been proposed, and of these the
cloverleaf model of Holley is the most widely accepted.
Transfer RNA (tRNA) is an essential component of the protein synthesis
reaction. There are at least twenty different kinds of tRNA in the cell1 and each
one serves as the carrier of a specific amino acid to the site of translation. tRNA’s
are L-shaped molecules.
The amino acid is attached to one end and the other end consists of three
anticodon nucleotides. The anticodon pairs with a codon in messenger RNA
(mRNA) ensuring that the correct amino acid is incorporated into the growing
polypeptide chain. The L-shaped tRNA is formed from a small single-stranded
RNA molecule that folds into the proper conformation. Four different regions of
double-stranded RNA are formed during the folding process (Refer Figure 3.3).

Fig. 3.3 Double-stranded RNA

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RNA: Structure and Types The two ends of the molecule form the acceptor stem region where the
amino acid is attached. The anticodon is an exposed single-stranded region in a
loop at the end of the anticodon arm. The two other stem/loop structures are
named after the modified nucleotides that are found in those parts of the molecule.
NOTES
The D arm contains dihydrouridylate residues while the TψC arm contains a
ribothymidylate residue (T), a pseudouridylate residue (ψ) and a Cytidylate (C)
residue in that order. All tRNA’s have a similar TψC sequence. The variable arm
is variable, just as you would expect. In some tRNA’s it is barely noticable while
in others it is the largest arm. tRNA’s are usually drawn in the ‘cloverleaf’ form to
emphasize the base-pairs in the secondary structure (Refer Figure 3.4).

Fig. 3.4 Cover Leaf Model of tRNA

Unusual Bases in tRNA

In addition to the usual bases A, U, G and C, tRNA contain a number of unusual
bases, and in this respect differs from mRNA and rRNA. The unusual bases of
tRNA account for 15-20% of the total RNA of the cell. Most of the unusual bases
are formed by methylation (addition of -CHa or methyl group to the usual bases),
for example Cytosine and Guanine on Methylation yield Methylcytosine and Methyl/
Guanine, respectively. Precursor tRNA molecules transcribed on the DNA
template contains the usual bases. These are then modified to unusual bases.
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 The unusual bases are important because they protect the tRNA molecule RNA: Structure and Types

against degradation by RNase. This protection is necessary because RNA is found

floating freely in the cell. Some of the unusual bases of tRNA are Methyl Guanine
(GMe), DiMethylguanine(GMe2), MEthylcytosine (Me), RiboThymine (T),
pseudouridine (ψ), DiHydrouridine (DHU, H2U, UH2), Inosine (I) and NOTES
Methylinosine (IMe, MeI). In general, organisms high in the evolutionary scale
contain more modified bases than lower organisms.
Classification of tRNA
A Study of different tRNAs shows that the structure of the acceptor stem, the
anticodon arm and the TøC arm are constant. The differences in the tRNAs lie in
the D arm and the variable arm. Based on the differences in these two variable
regions, three classes of tRNA have been recognized.
Class I (D4-V4-5), with 4 base pairs in the D stem and 4-5 bases in the variable
loop.
Class II (DS-V4-5), with 3 base pairs in the D stem and 4-5 bases pairs in the
variable loop.
Class III (D3-VN), with 3 base pairs in the D stem and a large variable arm.
A simpler classification based only on the variable arm recognizes two types of
tRNA.
Class I with 4-5 bases in the variable loop.
Class II with a large variable arm of 13-21 bases.
Tertiary Structure of Transfer - tRNA
Electron density maps have revealed that tRNA has a tertiary structure. This
structure is due to hydrogen bonds
• Between bases.
• Between bases and ribose phosphate backbone.
• Between the backbone residues. (The hydrogen bonding in the double helical
stem regions of the tRNA molecular are considered to be in the secondary
structure).
Initiator Transfer RNA - tRNA
The starting amino acid in eukaryote protein synthesis is methionine, while in
prokaryotes it is N-formyl methionine. The tRNA molecule3 specific for these
two amino acids are methionyl tRNA (tRNAmet) and N-formyl- methionyl IRNA
(tRNAfmet) respectively. These tRNAs are called initiator tRNAs, because they
initiate protein synthesis. Initiator tRNAs have certain features which distinguish
them from other tRNAs, and the initiator tRNAs of prokaryotes’ and eukaryotes
also differ. In most prokaryotes the 5' terminal nucleoside is C. It has opposite it
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RNA: Structure and Types (i.e., in the fifth position from the 3' end) an A nucleotide. There is no Watson-
Crick base pairing between the two. In the blue green ‘alga’ Anacystis nidulans,
however, the fifth nucleotide from the 3' end is C.
In eukaryotes there is an A.U base pair at the acceptor stem. As noted
NOTES
previously, prokaryotes use tRNAf-met for initiation of protein synthesis, while
eukaryotes use tRNAmet. The prokaryote Halo bacterium cutirubrum is, however,
reported to initiate protein synthesis with tRNA met and has an A.U base pair at
the end of the accept or stem. In these respects it resembles eukaryotes The D
loop of prokaryote initiator tRNAs contains an A11, U24 base pair. All other
tRNAs have a Y11, R24 base pair. Eukaryotic cytoplasmic initiator tRNAs have
AU or AU instead of Tψ in the TψC loop. Also, in eukaryotes instead of a
pyrimidine nucleotide (Y) there is A at the 3' end of the TψC loop. In some
eukaryotic cytoplasmic initiator tRNAs the anticodon sequence CAU is preceeded
by C instead of U as in all other tRNAs.
In prokaryotes the purine nucleotide following C in the TψC loop is A,
while in eukaryotes it is G. In tRNA fmet the nucleotide adjacent to the 3' side of
the anticodon triplet is adenosine while in tRNA met it is alkylated adenosine.
Specificity of Tranfer RNA - tRNA
Two important steps in translation during protein synthesis are the activation of
amino acids and the transfer of amino acids to tRNAs. Each amino acid has a
specific activating enzyme tRNA aminoacyl synthetase. Thus there are 20 different
tRNA aminoacyl synthetases for the 20 common amino acids found in proteins.
Some tRNA synthetases can activate more than one amino acid, i.e. they
show only a limited substrate specificity. Thus isoleucine tRNA synthetase can
also activate L valine, and valine tRNA synthetase can also react with threonine.
The enzymes, however, recognize only a specific set of, tRNAs as substratesL
isolecine tRNA synthetase recognizes only tRNAileu and valine tRNA synthetase
recognizes only tRNAval. Thus specificity is involved at two stages, activation of
the amino acid and transfer of the amino acid to tRNA. Another group of enzymes,
the tRNA aminoacyl transferases catalyse the transfer of an amino acid from the
amino acid - tRNA complex to specific acceptor molecules.

Check Your Progress

1. Define RNA.
2. How do cellular organisms use messenger RNA (mRNA)?
3. Name the types of cellular RNA.
4. What is tRNA?
5. How many types of steps are there in translation during protein synthesis?

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 RNA: Structure and Types
3.3 ANSWERS TO CHECK YOUR PROGRESS
QUESTIONS

1. RiboNucleic Acid (RNA) is a polymeric molecule essential in various NOTES

biological roles in coding, decoding, regulation and expression of genes.
2. Cellular organisms use messenger RNA (mRNA) to convey genetic
information (using the nitrogenous bases of Guanine, Uracil, Adenine,
and Cytosine, denoted by the letters G, U, A, and C) that directs synthesis
of specific proteins
3. Three types of cellular RNA have been distinguished:
• Messenger RNA (mRNA) or template RNA
• Ribosomal RNA (rRNA)
• Soluble RNA (sRNA) or transfer RNA (tRNA)
4. Transfer RNA (tRNA) is an essential component of the protein synthesis
reaction. There are at least twenty different kinds of tRNA in the cell and
each one serves as the carrier of a specific amino acid to the site of translation.
tRNA’s are L-shaped molecules.
5. Two important steps in translation during protein synthesis are the activation
of amino acids and the transfer of amino acids to tRNAs. Each amino acid
has a specific activating enzyme tRNA aminoacyl synthetase. Thus there
are 20 different tRNA aminoacyl synthetases for the 20 common amino
acids found in proteins.

3.4 SUMMARY

• RiboNucleic Acid (RNA) is a polymeric molecule essential in various

biological roles in coding, decoding, regulation and expression of genes.
• RNA and DNA are nucleic acids, and, along with lipids, proteins and
carbohydrates, constitute the four major macromolecules essential for all
known forms of life.
• Like DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it
is more often found in nature as a single-strand folded onto itself, rather
than a paired double-strand.
• Cellular organisms use messenger RNA (mRNA) to convey genetic
information (using the nitrogenous bases of Guanine, Uracil, Adenine,
and Cytosine, denoted by the letters G, U, A, and C) that directs synthesis
of specific proteins.
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RNA: Structure and Types • DNA to structures called ribosomes. These ribosomes are made from
proteins and ribosomal RNAs, which come together to form a molecular
machine that can read messenger RNAs and translate the information they
carry into proteins.
NOTES
• There are many RNAs with other roles – in particular regulating which
genes are expressed, but also as the genomes of most viruses.
• Ribose Nucleic Acids Most cellular RNA is single stranded, although some
viruses have double stranded RNA.
• The single RNA strand is folded upon itself, either entirely or in certain
regions. In the folded region a majority of the bases are complementary
and are joined by hydrogen bonds. This helps in the stability of the molecule.
• An important structural feature of RNA that distinguishes it from DNA is
the presence of a hydroxyl group at the 2' position of the ribose sugar.
• The presence of hydroxyl functional group causes the helix to adopt the A-
form geometry rather than the B-form most commonly observed in DNA.
• Ribosomal and transfer RNA comprise about 98% of all RNA. All three
forms of RNA are made on a DNA template.
• Transfer RNA and messenger RNA are synthesized on DNA templates of
the chromosomes, while ribosomal RNA is derived from nucleolar DNA.
• The three types of RNA are synthesized during different stages in early
development. Most of the RNA synthesized during cleavage is mRNA.
Synthesis of tRNA occurs at the end or cleavage, and rRNA synthesis
begins during gastrulation.
• Ribosomal RNA contains the four major RNA bases with a slight degree of
methylation, and shows differences in the relative proportions of the bases
between species. Its molecules appear to be single polynucleotide strands
which are unbranched and flexible.
• At low ionic strength rRNA behaves as a random coil, but with increasing
ionic strength the molecule shows helical regions produced by base pairing
between Adenine and Uracil and Guanine and Cytosine.

3.5 KEY WORDS

• RNA: RiboNucleic Acid or RNA, a nucleic acid present in all living cells.
Its principal role is to act as a messenger carrying instructions from DNA
for controlling the synthesis of proteins, although in some viruses RNA rather
than DNA carries the genetic information.
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 • Transfer RNA: Transfer RNA (tRNA) is an essential component of the RNA: Structure and Types

protein synthesis reaction.

3.6 SELF ASSESSMENT QUESTIONS AND NOTES

EXERCISES

Short Answer Questions

1. Write a short note on RNA.
2. What is rRNA?
3. Explain the structure of mRNA.
4. Explain with the help of diagram cover leaf model of tRNA.
Long Answer Questions
1. Briefly discuss about the RNA giving its significance and types.
2. Explain about chemical structure of RNA.
3. Discuss about the three types of cellular RNA.
4. Write a note on structure of mRNA.
5. Distinguish between mRNA, rRNA and tRNA.

3.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.

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RNA: Structure and Types McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
NOTES
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 Process of Transcription
BLOCK - II in Prokaryotes
PROTEIN SYNTHESIS & CLONING VECTORS

NOTES
UNIT 4 PROCESS OF
TRANSCRIPTION IN
PROKARYOTES
Structure
4.0 Introduction
4.1 Objectives
4.2 Process of Transcription in Prokaryotes
4.2.1 Process of Transcription
4.2.2 Mechanism of Transcription
4.2.3 Prokaryotic vs. Eukaryotic Transcription
4.3 Answers to Check Your Progress Questions
4.4 Summary
4.5 Key Words
4.6 Self Assessment Questions and Exercises
4.7 Further Readings

4.0 INTRODUCTION

Transcription, is the synthesis of RNA from DNA. Genetic information flows from
DNA into protein, the substance that gives an organism its form. This flow of
information occurs through the sequential processes of transcription (DNA to RNA)
and translation (RNA to protein).
During transcription, only one strand of DNA is usually copied. This is called
the template strand, and the RNA molecules produced are single stranded
messenger RNAs (mRNAs). The DNA strand that would correspond to
the mRNA is called the coding or sense strand. In eukaryotes (organisms that
possess a nucleus) the initial product of transcription is called a pre-mRNA. Pre-
mRNA is extensively edited through splicing before the mature mRNA is produced
and ready for translation by the ribosome, the cellular organelle that serves as the
site of protein synthesis. Transcription of any one gene takes place at the
chromosomal location of that gene, which is a relatively short segment of
the chromosome. The active transcription of a gene depends on the need for the
activity of that particular gene in a specific cell or tissue or at a given time.
The stretch of DNA transcribed into an RNA molecule is called a transcription
unit and encodes at least one gene. If the gene encodes a protein, the transcription
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Process of Transcription produces messenger RNA (mRNA); the mRNA, in turn, serves as a template for
in Prokaryotes
the protein’s synthesis through translation. Alternatively, the transcribed gene may
encode for non-coding RNA, such as microRNA, ribosomal RNA (rRNA),
transfer RNA (tRNA), or enzymatic RNA molecules called ribozymes. Overall,
NOTES
RNA helps synthesize, regulate, and process proteins; it therefore plays a
fundamental role in performing functions within a cell.
In this unit, you will study about transcription process in Prokaryotes,
elongation of RNA polymerase, mechanism of transcription, process of
transcription, differentiation between Prokaryotic and Eukaryotic transcriptions,
and how to detect the process of transcription.

4.1 OBJECTIVES

After going through this unit, you will be able to:

• Understand the what transcription is
• Discuss the mechanism of transcription
• Explain the process of transcription
• Differentiate between Prokaryotic and Eukaryotic transcriptions
• Analyse how to detect the process of transcription

4.2 PROCESS OF TRANSCRIPTION IN

PROKARYOTES

Transcription is the first step of DNA based gene expression, in which a particular
segment of DNA is copied into RNA (especially mRNA) by the enzyme RNA
polymerase. Both DNA and RNA are nucleic acids, which use base pairs of
nucleotides as a complementary language. During transcription, a DNA sequence
is read by an RNA polymerase, which produces a complementary, antiparallel
RNA strand called a primary transcript.
Meaning of Transcription
• Transcription is the process through which a DNA sequence is enzymatically
copied or duplicated by an RNA polymerase to produce a complementary
RNA.
• The synthesis or generation of RNA from a single strand of a DNA molecule
in the presence of enzyme RNA polymerase is called transcription.
• In other words, the process of formation of a messenger RNA molecule
using a DNA molecule is known as transcription. The studies that laid
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 the foundation for work that was carried out in the complex eukaryotes Process of Transcription
in Prokaryotes
came from the work initially performed on prokaryotes like bacterium,
Escherichia coli.
History and Background of Transcription NOTES

• In 1956 it was established that RNA could be synthesised in vitro using

RNA polymerase in vitro in laboratories but the properties of this RNA so
synthesised suggested that the presence of an additional factor was required
to terminate transcription correctly.
• Finally in 1972, the existence of this terminating enzyme was first proven by
Walter Fiers.
• Roger D. Kornberg was bestowed upon with the Nobel Prize in Chemistry
for his studies of the ‘Molecular Basis of Eukaryotic Transcription’
in 2006.
4.2.1 Process of Transcription

The main aspects relating to transcriptions are listed below:

Synthesis

• RNA is synthesized from a DNA template which is further subjected to

processing to form messenger RNA (mRNA), which is then used for synthesis
of a protein.
• Due to the synthesised RNA’s property of carrying a genetic message or
information from the DNA to the protein-synthesizing machinery of the cell
it is termed as messenger RNA (mRNA).
• The main distinguishing factor among others between RNA and DNA
sequence is the presence of U, or Uracil in RNA in place of the T, or
Thymine of DNA.
Template Used

• The synthesis of RNA occurs from a single strand of the DNA molecule.
The segment of DNA that is transcribed into an RNA molecule is termed
as a transcription unit which in turn further codes the sequence that is
translated into protein. It is also involved in direction and regulation of
protein synthesis.
• Template strand is that DNA strand which is used in RNA synthesis and it is
named so because it acts as the template for ordering the sequence of
nucleotides in an RNA transcript.
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Process of Transcription • Coding strand is that DNA strand which does not take part in DNA synthesis
in Prokaryotes
and has the same nucleotide sequence as that of the newly created RNA
transcript.
NOTES Enzyme Involved
• RNA polymerase is the enzyme that carries out transcription.
• It comprises of four kinds of polypeptides, namely α, β, β’ and σ, which
are bounded together to form a complex known as holoenzyme.
Genetic Information Copied
In this process, the genetic information coded in DNA is transcribed or copied
into a molecule of RNA.
First Step
• Gene expression comprises of two major steps namely transcription and
translation.
• Therefore the first step in the process of gene regulation or protein synthesis
is transcription.
Direction of Synthesis

• RNA is synthesized in the direction 5´ → 3´.

• RNA polymerase reads the DNA template strand in 3´ → 5´ direction and
the new RNA strand is synthesized in the 5´→ 3´ direction.
• RNA polymerase after binding to the 3´ end of a gene (promoter) on the
DNA template strand starts to travel toward the 5´ end.
4.2.2 Mechanism of Transcription
The mechanism of transcription consists of three major phases or stages namely:
• Initiation
• Elongation
• Termination
These are briefly discussed as follows:
Initiation
• Transcription initiates as the RNA polymerase binds to the promoter in
DNA in bacteria.
• The RNA polymerase being the core enzyme consists of five subunits: 2α
subunits, 1β subunit, 1β´ subunit, and 1σ subunit.
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 • At the beginning of initiation, the core enzyme is associated with a sigma Process of Transcription
in Prokaryotes
factor (number 70) that helps in finding the appropriate – 35 and – 10 base
pairs downstream of promoter sequences.
The initiation consists of the following steps: NOTES
• Formation of holoenzyme as a result of binding of RNA Polymerase (RNAP)
to one of several specificity factors the formation of holoenzyme facilitates
ease of recognition and binding to’ specific promoter regions in the DNA.
At this point, the DNA is double-stranded (‘closed’). This holoenzyme/
wound-DNA structure is known as the closed complex.
• Now the formation of open complex occurs which proceeds via the unwinding
of DNA into a single-stranded (‘open’) structure in the vicinity of the
initiation site (defined as + 1). This holoenzyme/unwound-DNA structure
so formed is known as the open complex.
• Although the transcription of the DNA is done by RNA but production of
about 10 abortive (short, non-productive) transcripts also occurs. These
abortive transcripts are unable to leave the RNA polymerase as the exit
channel is blocked by the σ-factor.
• The dissociation of σ-factor from the holoenzyme precedes elongation.
Formation of most transcripts occurs using Adenosine-5´ -TriPhosphate
(ATP) and sometimes using Guanosine-5´ -TriPhosphate (GTP) (Purine
Nucleoside Triphosphates) at the +1 site. However Uridine-5´- TriPhosphate
(UTP) and Cytidine-5´-TriPhosphate (CTP) (Pyrimidine Nucleoside
Triphosphates) are not favoured at the initiation site.
Elongation
• Transcription occurs using only one strand of DNA (called template strand
or non-coding strand) as a template and as it proceeds, RNA polymerase
travels the template strand and uses base pairing complementarity mechanism
to create an RNA copy.
• Although RNA polymerase travels the template strand from 3´ → 5´, the
coding (non-template) strand is generally used as the reference point, and
hence transcription is said to proceed in the direction 5´ → 3´.
• As a result an RNA molecule from 5´-3´ is produced which resembles the
coding strand exactly(except for the presence of thymine instead of uracil,
and the nucleotides are composed of a ribose (5-carbon) sugar as opposed
to DNA which has deoxyribose (one less oxygen atom) in its sugar-phosphate
backbone).
• The ‘abortive initiation cycle’ marks the beginning of elongation in case
of prokaryotes. This cycle involves the synthesis mRNA fragments 2-12
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Process of Transcription nucleotides long via RNA polymerase occurs. This process continues to
in Prokaryotes
occur till the rearrangement of the σ factor which results in the formation of
transcription elongation complex (which gives a 35 bp (base pair) moving
footprint). The σ-factor is released before 80 nucleotides of mRNA are
NOTES
formed.
Termination
In prokaryotes, there are two different modes of transcription termination as
described below:
• Rho-Independent
• Rho-Dependent
These are briefly discussed below:
Rho-Independent Termination

• Also called as intrinsic transcription termination.

• During this process the terminator sequences present within an RNA signal
the RNA polymerase to stop.
• The terminator sequence is usually a palindromic sequence that results in
the formation of a stem-loop hairpin structure which leads to the dissociation
of the RNAP from the DNA template.
In the Rho-independent transcription termination, RNA transcription ceases as
the newly synthesized RNA molecule forms a G-C rich hairpin loop, followed by
a run of U’s, that makes it detached from the DNA template.
Rho-Dependent Termination
• Unlike in the above case in, ‘Rho-Dependent’ type of termination, there
is a requirement of a protein factor named ‘Rho’ [r-factor] to stop RNA
synthesis at certain specific sites. This protein binds at a Rho utilisation site
on the nascent RNA strand and traverses along the mRNA towards the
RNA polymerase.
• Dissociation of RNAP from the DNA occurs as r-factor reaches the RNAP
thereby effectively terminating transcription. In other words, it leads to the
destabilization of the interaction between the template and the mRNA,
thereby resulting in the release of the newly synthesized mRNA from the
elongation complex.

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 Process of Transcription
in Prokaryotes

NOTES

Fig. 4.1 Process of Transcription

4.2.3 Prokaryotic vs. Eukaryotic Transcription

Although the process of transcription is the same in both eukaryotes and
prokaryotes in several aspects but there are some notable differences in
transcription of these two groups which are discussed below.
• Transcription in eukaryotes is much more complex than in prokaryotes.
• Transcription and translation take place separately in nucleus and cytoplasm
respectively in eukaryotes as opposed to the case in prokaryotes where
both processes take place simultaneously in the cytoplasm.
• The eukaryotic mRNA needs modification before taking part in protein
synthesis as it contains introns whereas prokaryotic mRNA does not require
any such modification.
• In eukaryotes the nucleus is present in DNA whereas in prokaryotes it is
present in the cytoplasm.

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Process of Transcription Table 4.1 Comparative Analysis of Difference in
in Prokaryotes Prokaryotic vs. Eukaryotic Transcription

S. No Prokaryotes Eukaryotes
NOTES 1 Transcription and Translation occurs Transcription and Translation occurs
simultaneously. separately.
2 The whole process is simple. The whole process is very
complicated.
3 mRNA does not undergoes any mRNA undergoes a lot of
modification. modifications.
4 RNA polymerase 2 α subunits, RNA polymerase I, II and III are
1 β subunit, 1 β’ subunit, and 1 σ subunit involved in the process.
are involved in the process.
5 The mechanism of termination is well The mechanism of termination is not
known. clear.
6 Transcription occurs in cytoplasm. Transcription occurs in nucleus.
7 DNA is circular in shape. DNA is linear in shape.

Detection of Transcription in Prokaryotes

Transcription can be measured and detected in many ways.
The commonly used methods of detecting transcription are listed below:
• The relative abundance of newly formed transcripts is measured by Nuclear
Run-on assay.
• The detection of active transcription sites is done by RNAse protection
assay and ChIP-Chip of RNAP.
• The absolute abundance of total or nuclear RNA levels (which may differ
from transcription rates) are measured by RT-PCR.
• The relative abundance of the global total or nuclear RNA levels are
measured by DNA microarrays.
• The presence of a transcript is detected by in-situ hybridization.

Check Your Progress

1. What is transcription?
2. Explain synthesis that occur in transcription.
3. What are the enzyme involved in transcription?
4. Name the phases of mechanism of transcription.
5. Explain briefly the initiation process.

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 Process of Transcription
4.3 ANSWERS TO CHECK YOUR PROGRESS in Prokaryotes

QUESTIONS

1. Transcription is the process through which a DNA sequence is enzymatically NOTES

copied or duplicated by an RNA polymerase to produce a complementary
RNA.
2. Following are the key highlights of synthesis that occur in transcription:
• RNA is synthesized from a DNA template which is further subjected to
processing to form messenger RNA [mRNA], which is then used for
synthesis of a protein.
• Due to the synthesised RNA’s property of carrying a genetic message
or information from the DNA to the protein-synthesizing machinery of
the cell it is termed as messenger RNA (mRNA).
• The main distinguishing factor among others between RNA and DNA
sequence is the presence of U, or uracil in RNA in place of the T, or
thymine of DNA.
3. Enzyme involved in transcription are as follows:
• RNA polymerase is the enzyme that carries out transcription.
• It comprises of four kinds of polypeptides, namely α, β, β’ and σ,
which are bound together to form a complex known as holoenzyme.
4. The mechanism of transcription consists of three major phases or stages
namely:
• Initiation
• Elongation
5. Following are the steps that are involved in initiation process:
• Transcription initiates as the RNA polymerase binds to the promoter in
DNA in bacteria.
• The RNA polymerase being the core enzyme consists of five subunits:
2α subunits, 1β subunit, 1β’ subunit, and 1σ subunit.
• At the beginning of initiation, the core enzyme is associated with a sigma
factor (number 70) that helps in finding the appropriate –35 and – 10
base pairs downstream of promoter sequences.

4.4 SUMMARY

• Transcription is the process through which a DNA sequence is enzymatically

copied or duplicated by an RNA polymerase to produce a complementary
RNA.
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Process of Transcription • The mechanism of transcription consists of three major phases or stages
in Prokaryotes
namely: Initiation, Elongation and Termination.
• Transcription initiates as the RNA polymerase binds to the promoter in
NOTES DNA in bacteria.
• The RNA polymerase being the core enzyme consists of five subunits: 2α
subunits, 1β subunit, 1β’ subunit, and 1σ subunit.
• Transcription occurs using only one strand of DNA [called template strand
or non-coding strand] as a template and as it proceeds, RNA polymerase
travels the template strand and uses base pairing complementarity mechanism
to create an RNA copy.
• The synthesis of RNA occurs from a single strand of the DNA molecule.
The segment of DNA that is transcribed into an RNA molecule is termed as
a transcription unit which in turn further codes the sequence that is translated
into protein. It is also involved in direction and regulation of protein synthesis.
• Template strand is that DNA strand which is used in RNA synthesis and it
is named so because it acts as the template for ordering the sequence of
nucleotides in an RNA transcript.
• Coding strand is that DNA strand which does not take part in DNA synthesis
and has the same nucleotide sequence as that of the newly created RNA
transcript.
• In prokaryotes, there are two different modes of transcription termination
namely (i) Rho-Independent and (ii) Rho-Dependent.
• Transcription in eukaryotes is much more complex than in prokaryotes.
• The eukaryotic mRNA needs modification before taking part in protein
synthesis as it contains introns whereas prokaryotic mRNA does not require
any such modification.
• Transcription in eukaryotes is much more complex than in prokaryotes.
• Transcription and translation take place separately in nucleus and cytoplasm
respectively in eukaryotes as opposed to the case in prokaryotes where
both processes take place simultaneously in the cytoplasm.
• The eukaryotic mRNA needs modification before taking part in protein
synthesis as it contains introns whereas prokaryotic mRNA does not require
any such modification.
• In eukaryotes the nucleus is present in DNA whereas in prokaryotes it is
present in the cytoplasm.
• The relative abundance of newly formed transcripts is measured by Nuclear
Run-on assay.
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 • The detection of active transcription sites is done by RNAse protection Process of Transcription
in Prokaryotes
assay and ChIP-Chip of RNAP.
• The absolute abundance of total or nuclear RNA levels (which may differ
from transcription rates) are measured by RT-PCR.
NOTES
• The relative abundance of the global total or nuclear RNA levels are
measured by DNA microarrays.
• The presence of a transcript is detected by in situ hybridization.

4.5 KEY WORDS

• Holoenzyme: A compound formed by the combination of enzyme and a

coenzyme
• Template strand: It is the strand which is used by DNA polymerase or
RNA polymerase to attach complementary bases during DNA replication
or RNA transcription
• Coding strand: The coding strand is the DNA strand whose base sequence
corresponds to the base sequence of the RNA transcript produced
• In-situ hybridization: It is a technique used to detect particular target DNA
or RNA molecule within a complex of DNA and RNA molecules.
• Microarray: It is a technique used to study the expression levels of large
number of genes simultaneously.

4.6 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is transcription?
2. Explain the mechanism of transcription.
3. What is initiation?
4. Define elongation.
5. Write a short note on termination.
6. Distinguish between Prokaryotic and Eukaryotic transcriptions.
Long Answer Questions
1. Explain the process of transcription
2. Discuss briefly the mechanism of transcription.
3. Discuss in detail the process of initiation, elongation and termination involved
in the process of transcription.
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Process of Transcription 4. Differentiate between the Prokaryotic and Eukaryotic process of
in Prokaryotes
transcriptions.
5. List the methods that can be used for detecting the process of transcription.
NOTES
4.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 RNA Processing

UNIT 5 RNA PROCESSING

Structure NOTES
5.0 Introduction
5.1 Objectives
5.2 RNA Processing
5.3 Answers to Check Your Progress Questions
5.4 Summary
5.5 Key Words
5.6 Self Assessment Questions and Exercises
5.7 Further Readings

5.0 INTRODUCTION

All classes of RNA transcripts must be processed into mature species. The reactions
include several types: Nucleolytic cleavage, as in the separation of the mature
rRNA species from the primary transcript of RNA polymerase I action; Chain
extension (non template directed), as in the synthesis or regeneration of the common
CCA sequence at the 3´ end of transfer RNAs or of PolyA at the 3´ end of
mRNAs; and Nucleotide modification, for example, the synthesis of methylated
nucleotides in tRNA or rRNA. These reactions are a feature of both prokaryotic
and eukaryotic gene expression, and the biological consequences are diverse. For
example, modified nucleotides can affect the way in which a tRNA recognizes
different codons.
Messenger RNA processing is complex, especially in eukaryotes.
Prokaryotic mRNA processing is relatively unimportant in regulating gene
expression. The chief function of prokaryotic mRNA seems to be to regulate
stability. The terminator stem and loop stabilizes mRNA against nucleolytic
degradation, and in some cases, removal of this structure destabilizes mRNA so
that it is transcribed less efficiently.
Eukaryotic mRNA processing is much more complex and has many
consequences for gene expression. Most obviously, many eukaryotic genes
contain introns, which are found in the primary transcript. For the mRNA to be
translated into a useful protein, some way to remove them from the transcript and
still preserve the coding sequence of the mature messenger RNA must exist.
Additionally, the 5´ ends of eukaryotic mRNAs contain a specialized cap structure
and a 3´ polyA tail, and these are not encoded by the DNA template. Many of
these reactions involve small nuclear ribonucleoprotein particles (snRNPs or snurps)
as essential components. Other submicroscopic structures in the nucleus are
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RNA Processing
essential for processing and transport of the mature RNAs from the nucleus into
the cytoplasm.
In this unit, you will study about RNA processing (post transcriptional
NOTES modifications), inhibitors of transcription and reverse transcription in detail.

5.1 OBJECTIVES

After going through this unit, you will be able to:

• Understand the processing of mRNA
• Discuss the processing of rRNA and tRNA
• Explain the types of inhibitors of transcription
• Understand the process of reverse transcription

5.2 RNA PROCESSING

In the process of gene expression, transcription is referred to as the process in

which information from a gene is used to construct a functional product, such as a
protein. The main purpose of the process of transcription is to make a RNA copy
of a gene’s DNA sequence. For a protein-coding gene, the RNA copy, or
transcript, carries the information needed to build a polypeptide. Eukaryotic
transcripts often need to undergo some processing steps before translation into
proteins.
Processing of Eukaryotic mRNA

RNA processing is a transformation which almost all mRNA molecules undergo

after synthesis except for prokaryotic mRNA. In prokaryotes, translation at 5´ -
end of prokaryotic mRNA starts while the 3´ -end is still being synthesised. Although
both prokaryotic and eukaryotic tRNAs and rRNAs undergo processing,
eukaryotic mRNAs are processed the maximum. Unlike in the case of prokaryotic
mRNA, all other kinds of RNA undergo processing immediately after synthesis.
Primary transcription or precursor mRNA is the newly synthesised mRNA
which is quite different from the mRNA that takes part in protein synthesis.
Processing of mRNA is brought about by significant changes in precursor mRNA.
Various changes that lead to the formation of mature mRNA include removal of
non-coding regions by splicing and certain medications at the 5´ -end 3´ -end of
mRNA (Refer Figure 5.1).

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 RNA Processing

NOTES

Fig. 5.1 Processing of mRNA via Splicing

5´ -End Capping
This is a process in which a nucleotide is added to the 5´ -end of hnRNA, in the
following steps:
• Addition of a cap of 7-methylguanosine (m7G or 7MeG) immediately after
or during transcription at 5´ -end of mRNA.
• Addition of this cap in reverse orientation of 5´ → 5´ instead of normal
5´ → 3´ orientation giving a 5-5´ triphosphate bridge.
• Addition of a methyl group to the 7th position of terminal guanine. Enzyme
guanylyl transferase is involved in the catalysis of this reaction.
• Significance of capping: The cap confers stability to mRNA as due to the
inability exonuclease to act on it.
3´ -Cleavage and Polyadenylation
It is a process in which addition of a tail comprising sequence of adenine (adenylic
acid) at the 3´-end of mRNA occurs immediately after transcription. This tail
consisting of 80-200 adenine nucleotides confers stability to the mRNA and is
called as Poly (A) tail. Enzyme Poly (A) Polymerase or PAP is involved in this
process of polyadenylation.
Recent discovery of coding sequences of most eukaryotic genes being split
into stretches of codons interrupted by stretches of non-coding sequences threw
light on structure of DNA as opposed to earlier believes of coding sequences if
DNA and amino acids of polypeptide chain being collinear.
• The coding sequences of DNA are known as exons which are intervened
by non-coding sequences called introns. These types of genes are called
split genes or interrupted genes.
• Gilbert coined the terms exons and introns. Introns usually being larger in
size than the exons constitute a large portion of the genome.
• Processing of mRNA occurs by a process of RNA splicing in which the
introns are excised out and removed.
• Splicing is followed by translation.

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RNA Processing Processing of tRNA
Following are the ways in which processing of tRNA occur (Refer Figure 5.2):
• Due to the large size of precursor tRNA as compared to mature tRNA
NOTES which comprises of only 80-90 nucleotides, tRNA undergoes extensive
processing.
• For instance, tRNATyr which is a tyrosine carrying tRNA consists of 350
nucleotides. The processing of this tRNA leads to removal the useless
sequences. Various enzymes like RNase D, RNase E, RNase F and RNase
P are involved in the removal of nucleotides from both 5´ and 3´ ends.
• Endonucleases are enzymes that also remove many sequences by cleaving
the phosphodiester link within a polynucleotide chain. Once the primary
transcript folds and forms peculiar stems and loop structure by
comprehensive complementary base pairing cleaving is done. RNase P is a
ribozyme.
• The enzyme tRNA nucleotidyl transferase adds the 5´ -CCA-3´ sequence
at 3´ -end of mature tRNA which leads to the generation of the mature
tRNA.
• Several unusual bases like pseudouridine (Ψ), 2- isopentenyladenosine
(2 ip A), 2-O-methylguanosine (2m G), 4-thiouridine (4 tµ), Ribothymidine,
dihyrouridine and inosine are formed by the modification of normal existing
bases A, G, C and U via enzymatic action.

Fig. 5.2 Figure Depicting (a) Precursor t-RNA (b) Mature t-RNA

rRNA Processing
rRNA processing in both prokaryotes and eukaryotes occurs by removal and/or
addition and/or modification of certain nucleotides which brings about changes in
the primary transcript. The removal of nucleotides is facilitated by exonucleases
and endonucleases.
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rRNA Processing in Prokaryotes RNA Processing

• In Escherichia coli there are various different operons in rRNA, each of

which contains one copy of 5S, 16S and 23S rRNA sequences.
• Moreover, various coding sequences of tRNA molecules are also present in NOTES
rRNA operons.
• These primary transcriptions are processed to produce both rRNA and
tRNA molecules. The cleavage of a continuous transcript of more than 5000
nucleotides yields all these molecules.
rRNA Processing in Eukaryotes
• The precursor rRNA in eukaryotes comprises of one copy of 18S coding
region, one copy of 5.8S and one copy of 28S coding region.
• The transcription of rRNA precursor takes place in the nucleolus which is
facilitated by RNA polymerase I.
• RNA polymerase III transcribes eukaryotic 5S rRNA from an unlinked
gene.
• rRNA molecules form secondary structures of several double stranded stems
by complementary base pairing and single stranded loops in both eukaryotes
and prokaryotes.

Fig. 5.3 Processing of rRNA

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RNA Processing • snoRNAs are small nucleolar RNAs which are required for the processing
of rRNA molecules in eukaryotes. These are present in the nucleolus where
rRNA is processed along with the ribosomes which also assemble in
nucleolus.
NOTES
• RNA molecules are present in the cells in the form of complexes which
result due to the combination of specific RNA molecules with specific
proteins. Theses RNA-protein complexes are known as ribonuclear proteins
or RNPs. The largest RNPs formed by rRNA molecules forming complexes
with specific ribosomal proteins are ribosomes. In Escherichia coli 25%
of the dry weight of the cell is due to ribosomes which consist of 10% of
total proteins and 80% of total RNA.
• In prokaryotes 70S ribosome consists of 30S and 50S subunits in which
the former has one copy of 16S rRNA and 21 different proteins while the
later has 23S rRNA, 5S rRNA and 31 different proteins.

Fig. 5.4 Types of rRNA Found in Prokaryotes and Eukaryotes

• In eukaryotes 80S ribosome comprises of 40S and 60S subunit in which

the former has 18S rRNA and 30 different proteins while the later has 28S
rRNA, 5.8S rRNA, 5.8S rRNA and 45 different proteins.
Inhibitors of Transcription
• Transcription inhibitors, as the name suggests, inhibit the process of
transcription.
• They are often used as antibiotics against and to curb the growth of
pathogenic bacteria and fungi.
• For instance rifampicin inhibits bacterial transcription of DNA into mRNA
by causing inhibition of DNA-dependent RNA polymerase by binding to
its beta-subunit.
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 • Another example includes hydroxyquinoline which is an antifungal RNA Processing

transcription inhibitor.
• Several other commonly used transcription inhibitors include Alpha-
Amanitin which works by binding to the largest subunits of RNA Polymerase NOTES
I and II.
Reverse Transcription
• Reverse transcription is the synthesis of DNA from RNA molecule in the
presence of enzyme reverse transcriptase. Temin and Baltimore were
awarded Nobel Prize in 1975 for reporting about reverse transcription in
1970. Reverse transcription is famously called Teminism after the scientist
who first reported it. Some viruses (such as HIV, the cause of AIDS), have
the potential to transcribe RNA into DNA.
• HIV has an RNA genome that is duplicated into DNA which can further be
merged with the DNA genome of the host cell. Reverse transcriptase is the
primary enzyme responsible for the generation of DNA from an RNA
template. In the case of HIV, it is reverse transcriptase which synthesizes a
complementary DNA strand (cDNA) to the viral RNA genome.
• An accessory enzyme, ribonuclease H works by digesting the RNA strand
while reverse transcriptase synthesizes a complementary strand of DNA
leading to the formation of a double helix DNA structure. This cDNA is
additionally integrated into the host cell’s genome via another enzyme namely
integrase which causes the host cell to produce viral proteins which
reassemble into new viral particles which eventually leads to the programmed
cell death (apoptosis) of the host cells.
Table 5.1 Table Depicting the Differences in the Process of
Transcription and Reverse Transcription

S.No Transcription Reverse Transcription

1 It occurs in both Prokaryotes It occurs in some
and Eukaryotes. viruses.
2 It helps in the synthesis of It helps in the synthesis
RNA from DNA. of DNA from RNA.
3 Transcriptase is the enzyme Reverse transcriptase is
involved in the process. the enzyme involved in
the process.
4 DNA is used as the template. RNA is used as the
template.

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RNA Processing

Check Your Progress

1. How a nucleotide is added to the 5´ -end of hnRNA?
NOTES 2. What are the steps included in processing of tRNA?
3. Define rRNA processing.
4. How does rRNA processing in Prokaryotes?

5.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. The is a process in which a nucleotide is added to the 5´ -end of hnRNA, in

the following steps:
• Addition of a cap of 7-methylguanosine (m7G or 7MeG) immediately
after or during transcription at 5´ -end of mRNA.
• Addition of this cap in reverse orientation of 5´ → 5´ instead of normal
5´ → 3´ orientation giving a 5-5´ triphosphate bridge.
• Addition of a methyl group to the 7th position of terminal guanine. Enzyme
guanylyl transferase is involved in the catalysis of this reaction.
• Significance of capping: The cap confers stability to mRNA as due to
the inability exonuclease to act on it.
2. Following steps occurs in processing of tRNA:
• Due to the large size of precursor tRNA as compared to mature tRNA
which comprises of only 80-90 nucleotides, tRNA undergoes extensive
processing.
• For instance, tRNATyr which is a tyrosine carrying tRNA consists of
350 nucleotides. The processing of this tRNA leads to removal the useless
sequences. Various enzymes like RNase D, RNase E, RNase F and
RNase P are involved in the removal of nucleotides from both 5´ and 3´
ends.
• Endonucleases are enzymes that also remove many sequences by
cleaving the phosphodiester link within a polynucleotide chain. Once
the primary transcript folds and forms peculiar stems and loop structure
by comprehensive complementary base pairing cleaving is done. RNase
P is a ribozyme.
• The enzyme tRNA nucleotidyl transferase adds the 5´-CCA-3´ sequence
at 3´-end of mature tRNA which leads to the generation of the mature
tRNA.
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 • Several unusual bases like pseudouridine (Ψ), 2- isopentenyladenosine RNA Processing

(2 ip A), 2-O-methylguanosine (2m G), 4-thiouridine (4 tµ),

Ribothymidine, dihyrouridine and inosine are formed by the modification
of normal existing bases A, G, C and U via enzymatic action.
NOTES
3. rRNA processing in both prokaryotes and eukaryotes occurs by removal
and/or addition and/or modification of certain nucleotides which brings about
changes in the primary transcript .The removal of nucleotides is facilitated
by exonucleases and endonucleases.
4. rRNA processing in Prokaryotes occur as below:
• In Escherichia coli there are various different operons in rRNA, each
of which contains one copy of 5S, 16S and 23S rRNA sequences.
• Moreover, various coding sequences of tRNA molecules are also present
in rRNA operons.
• These primary transcriptions are processed to produce both rRNA
and tRNA molecules. The cleavage of a continuous transcript of more
than 5000 nucleotides yields all these molecules.

5.4 SUMMARY

• Apart from prokaryotic mRNA all other kinds of RNAs are processed
immediately after synthesis.
• Eukaryotic mRNA is subjected to maximum processing.
• Processing of Eukaryotic mRNA includes newly synthesized mRNA known
as pre-mRNA, is quite different from the mRNA that takes part in protein
synthesis. This is due to the processing that it undergoes, for instance tailing
and capping. A cap of 7-methylguanosine (m7G or 7 Me G) is added at 5´
end of mRNA and a tail consisting of 80-200 adenine nucleotides called
Poly(A) tail is added at 3´-end.
• Non-coding regions or introns are removed via splicing and coding regions
or exons are ligated together.
• Both prokaryotic and eukaryotic tRNAs and rRNAs are subjected to
comprehensive processing.
• Pre-tRNA consists about 350 nucleotides out of which the useless ones are
discarded during processing.
• Mature tRNA consists of approximately 70 nucleotides.
• At the 3´ end CCA sequence is joined.
• The modification of normal bases A, G, C and U often give rise to various
unusual bases.
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RNA Processing • In both prokaryotes and eukaryotes, rRNA molecules form secondary
structures of several double stranded stems by complementary base pairing
and single stranded loops.
NOTES • Many proteins bind to the snrRNA molecules to form RiboNucleoProteins
(RNP) in the ribosomes. There are several small nuclear RNA molecules
called small nuclear RNAs (snRNA).
• Transcription inhibitors, as the name suggests, inhibit the process of
transcription.
• They are often used as antibiotics against and to curb the growth of
pathogenic bacteria and fungi.
• For instance rifampicin inhibits bacterial transcription of DNA into mRNA
by causing inhibition of DNA-dependent RNA polymerase by binding to
its beta-subunit.
• Reverse transcription is the synthesis of DNA from RNA molecule in the
presence of enzyme reverse transcriptase.
• Temin and Baltimore were awarded Nobel Prize in 1975 for reporting about
reverse transcription in 1970.
• Reverse transcription is famously called Teminism after the scientist who
first reported it.
• Some viruses (such as, HIV, the cause of AIDS), have the potential to
transcribe RNA into DNA.
• HIV has an RNA genome that is duplicated into DNA which can further be
merged with the DNA genome of the host cell.
• Reverse transcriptase is the primary enzyme responsible for the generation
of DNA from an RNA template.
• In the case of HIV, it is reverse transcriptase which synthesizes a
complementary DNA strand (cDNA) to the viral RNA genome.
• An accessory enzyme, ribonuclease H works by digesting the RNA strand
while reverse transcriptase synthesizes a complementary strand of DNA
leading to the formation of a double helix DNA structure.

5.5 KEY WORDS

• Transcription: Process of formation of RNA from DNA sequence.

• 7-methylguanosine: A cap like structure present at the 5end of mRNA.
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 • Exons: A part of gene that will ultimately form a part of final mature RNA. RNA Processing

• Introns: Non-coding DNA found between exons of a gene.

• Split genes: A gene that contains sections of coding portion like exon
interrupted by non-coding portion like introns. NOTES
• Reverse transcription: It is the process of obtaining DNA from RNA in
the presence of enzyme reverse transcriptase.

5.6 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is RNA processing?
2. What is 5´ -end capping?
3. How does processing of rRNA in Eukaryotes occurs?
4. Explain inhibitors of transcription.
5. What is reverse transcription?
Long Answer Questions
1. Discuss the process of mRNA processing in eukaryotes.
2. Explain the process of tRNA processing.
3. Differentiate between the processing of rRNA in Prokaryotes vs. Eukaryotes.
4. Discuss in detail the process of transcription and reverse transcription.
5. List and explain some inhibitors for process of transcription.

5.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
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RNA Processing Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
NOTES Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 Cloning Vectors

UNIT 6 CLONING VECTORS

Structure NOTES
6.0 Introduction
6.1 Objectives
6.2 Cloning Vectors
6.2.1 Plasmids as Cloning Vectors
6.2.2 Phage λ
6.2.3 Cosmid Vectors
6.2.4 Phagemids
6.2.5 Vectors Required for the Cloning of Larger DNA Segments
6.2.6 Yeast Artificial Chromosomes (YAC Vectors)
6.2.7 Vectors for the Preparation of ssDNA
6.2.8 Viral Vector – SV 40 and Adenovirus
6.3 Answers to Check Your Progress Questions
6.4 Summary
6.5 Key Words
6.6 Self Assessment Questions and Exercises
6.7 Further Readings

6.0 INTRODUCTION

A cloning vector is a small piece of DNA that can be stably maintained in an

organism, and into which a foreign DNA fragment can be inserted for
cloning purposes. The cloning vector may be DNA taken from a virus, the cell of
a higher organism, or it may be the plasmid of a bacterium. The vector therefore
contains features that allow for the convenient insertion or removal of a DNA
fragment to or from the vector, for example by treating the vector and the foreign
DNA with a restriction enzyme that cuts the DNA. DNA fragments thus generated
contain either blunt ends or overhangs known as sticky ends, and vector DNA
and foreign DNA with compatible ends can then be joined together by molecular
ligation. After a DNA fragment has been cloned into a cloning vector, it may be
further subcloned into another vector designed for more specific use.
There are many types of cloning vectors, but the most commonly used ones
are genetically engineered plasmids. Cloning is generally first performed using
Escherichia coli, and cloning vectors in Escherichia coli include plasmids,
bacteriophages (such as, phage λ), cosmids, and Bacterial Artificial Chromosomes
(BACs). Some DNA, however, cannot be stably maintained in Escherichia coli,
for example very large DNA fragments, and other organisms such as yeast may be
used. Cloning vectors in yeast include Yeast Artificial Chromosomes (YACs).
In this unit, you will study about cloning vectors – plasmids, cosmids,
phasmids, phagemids, expression vectors, plasmid vectors – pBR322 and
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Cloning Vectors pUC18, integrating shuttle vector –YAC vectors, viral vector – SV 40 and
adenovirus in detail.

NOTES 6.1 OBJECTIVES

After going through the unit, you will be able to:

• Analyse what cloning vectors are
• Explain specific features of vectors
• Discuss the different types of vectors like plasmid, cosmid, phasmids
• Understand the uses of vectors
• Describe viral vectors – SV 40 and adenovirus

6.2 CLONING VECTORS

A cloning vector is a small piece of DNA that can be stably maintained in an

organism, and into which a foreign DNA fragment can be inserted for cloning
purposes. The cloning vector may be DNA taken from a virus, the cell of a higher
organism, or it may be the plasmid of a bacterium. The vector therefore contains
features that allow for the convenient insertion or removal of a DNA fragment to
or from the vector, for example by treating the vector and the foreign DNA with a
restriction enzyme that cuts the DNA. DNA fragments thus generated contain
either blunt ends or overhangs known as sticky ends, and vector DNA and foreign
DNA with compatible ends can then be joined together by molecular ligation.
After a DNA fragment has been cloned into a cloning vector, it may be further
subcloned into another vector designed for more specific use.
There are many types of cloning vectors, but the most commonly used ones
are genetically engineered plasmids. Cloning is generally first performed using
Escherichia coli, and cloning vectors in Escherichia coli include plasmids,
bacteriophages (such as, Phage λ), cosmids, and Bacterial Artificial Chromosomes
(BACs). Some DNA, however, cannot be stably maintained in Escherichia coli,
for example very large DNA fragments, and other organisms, such as yeast may
be used. Cloning vectors in yeast include Yeast Artificial Chromosomes (YACs).
All commonly used cloning vectors in molecular biology have key features
necessary for their function, such as a suitable cloning site and selectable marker.
Others may have additional features specific to their use. For reason of ease and
convenience, cloning is often performed using Escherichia coli. Thus, the cloning
vectors used often have elements necessary for their propagation and maintenance
in Escherichia coli, such as a functional Origin Of Replication (OOR). The ColE1
origin of replication is found in many plasmids. Some vectors also include elements
that allow them to be maintained in another organism in addition to Escherichia
coli, and these vectors are called shuttle vector.
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6.2.1 Plasmids as Cloning Vectors Cloning Vectors

A cloning vector is DNA fragment derived from a virus or plasmid of bacterium or

cell of higher organism which can be inserted into a foreign DNA for the purpose
of cloning. It also has the ability to remain intact in the body of other organism. NOTES
Consequently, the cloning vector contains features that permit for the easy insertion
or removal of a DNA fragment to or from the vector.
Plasmids are extra chromosomal, double-stranded DNA molecules that
replicate autonomously within bacterial cells. Most of the plasmids are circular in
structure but linear plasmids have been found in some bacteria like Borrelia
burgdorferi and Streptomyces. The size of the naturally occurring plasmids ranges
from a few thousand base pairs to more than 100 kilo-bases (kb). Replication of
plasmids depends upon the same set of enzymes that are responsible for the
replication of chromosome of the host cells. Further, they are segregated to daughter
cells alongside the host chromosome during cell division assuring sustained
propagation of the plasmid through consecutive generations of the bacterial host
cell.
Plasmids generally contain genes that help the host cells. For instance, a
bacterial cell containing plasmids that encodes antibiotic inactivating enzymes can
replicate in an environment containing the antibiotic while other bacterium lacking
the drug-resistant plasmid failed to thrive.
Salient Features of Plasmids
• Plasmids may be single copy plasmids as they are present as one plasmid
DNA per bacterial cell.
• Plasmids may be multicopy plasmids as they are maintained as 10-12
genomes per host cell.
• Plasmids are also capable of increasing their copy number in the host cell
up to 100 copies under relaxed replication control.
Plasmids are the first vector that were developed for gene cloning experiments
and till date are widely used due to their versatile nature. However, to act as
cloning vectors, all plasmids should possess the following properties:
• They should be easily isolated from their host cells.
• They should contain single restriction site for at least one or more restriction
enzymes.
• Inserting a desirable piece of a linear foreign DNA at one end of these sites
should not modify the replication properties of plasmid.
• Plasmids can be easily reintroduced back into the host bacterial cells.
• Bacterial host cells carrying the plasmid with or without foreign insert should
be effortlessly selected or identified.
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Cloning Vectors Modifications of Plasmids
Most plasmids commonly used as cloning vector in molecular biology, replicate in
the bacterium Escherichia coli. These plasmids may not have all the features
NOTES required for uncomplicated DNA cloning and hence are genetically modified or
engineered to optimize their use as cloning vectors in DNA cloning.
Some of the Optimized characteristics of Plasmids:
• Length is shortened to easily work with plasmids; many such plasmids used
as cloning vectors are only 3 kb in length, much shorter than their original
lengths.
• Most plasmid used as cloning vectors contain little more than the necessary
nucleotide sequences essential for their use in DNA cloning: a drug-resistance
gene, a replication origin and a region in which exogenous foreign DNA
fragment can be inserted.
• Plasmids to be used as cloning vectors are also customized to increase their
copy number in host bacterial cells by the process of amplification.
• Plasmid vectors generally are modified to have a Multiple Cloning Sites
(MCSs). MCS is a short DNA sequence (for instance, it is around 2.8 kb
in case of PUC 19) carrying cleaving sites for several restriction
endonucleases. An MCS enhances the number of possible cloning
approaches available by widening the range of enzymes which can be utilized
to produce a restriction fragment apt for cloning.
pBR Vector Plasmids
pBR group of plasmids is the most widely used cloning vectors named after the
discoverer of these plasmids namely Bolivar and Rodriguez. Among pBR plasmids,
we will be discussing pBR322 (Refer Figure 6.1):
• It has been entirely sequenced through alteration of earlier plasmids of
Escherichia coli, pBR318 and pBR320.
• The pBR322 plasmid is about 4362 bp in length and possess an Origin Of
Replication (OOR), derived from a plasmid related to ColEI.
• It also possesses some genes which confers resistance to antibiotics, for
example ampicillin (AmpR) and tetracyclin (TetR).
• There are over 10 enzymes with unique cleavage sites on the pBR322
genome.
• The target sites of these enzymes lie within the tetracyclin resistant gene
(TetR). Six unique restriction sites also lie within the AmpR gene.
• Apart from cloning vector, it is widely used as a model system for the study
of prokaryotic transcription and translation as well as for observing the
effects of topological changes on DNA conformation.
• Cloning of foreign DNA in pBR322 with the aid of any one of these restriction
enzymes will lead to insertional inactivation of either the TetR or
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 AmpR markers. For instance: Cloning into Hindlll site of pBR322 generally Cloning Vectors

results in loss of tetracycline resistance. The recombinant plasmid with insert

will then allow the bacterial host cells to grow in the culture medium only in
presence of either ampicillin or tetracycline but not both. On the other hand,
plasmids lacking DNA insert, will be able to thrive in culture media containing NOTES
one or both the antibiotics. However, cloning can also occur in other unique
restriction sites which do not permit the easy selection of recombinants.
• In pBR322, the PstI site in the AmpR gene is mainly useful, because the 32
tetra-nucleotide extensions formed on digestion are ideal structures for the
enzyme terminal transferase.
• Over the years, numerous superior plasmid cloning vectors have been
constructed from pBR322 via inclusion of additional selected markers and
unique restriction sites. For instance, pBR325 plasmid encodes an additional
chloramphenicol resistance gene along with tetracycline and ampicillin
resistance and also carries a unique EcoRI site in the CmR gene.
• Another cloning vector pBR327 was derived from pBR322 by removal of
nucleotides from 1427 to 2516 thus reducing the size of vector. However,
pBR327 plasmids like pBR322 also possess two antibiotic resistance genes
namely AmpR and TetR.

Fig. 6.1 pBR322 Plasmid

pUC Vector Plasmids

pUC plasmids were named pUC as they were first prepared in University of
California.
pUC plasmids possess several characteristics as a vector for cloning like:
• Reduced size of pUC cloning vector can carry relatively large foreign DNA
inserts.
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Cloning Vectors • pUC18 (type of pUC plasmid) can replicate inside the bacterial host cell to
form about 500 copies per cell.
• It contains only one Ampicillin resistance gene.
NOTES • It contains an origin of replication that is generally derived from Escherichia
coli plasmid pBR322.
• pUC plasmids have a unique selection system that permits easy identification
between recombinant plasmids carrying a foreign insert and non-
recombinants. Plasmid cloning vector pUC18 contains a lac Z gene derived
from Escherichia coli. Within the lac region, a multiple cloning poly-linker
site is present which contains numerous sites for restriction endonucleases.
The lac gene is inactivated, when foreign DNA of appropriate length is
inserted in this region. Such recombinant plasmids form white colonies,
when transformed into the host lac negative (lac-) Escherichia coli strain
and grown in presence of IPTG (IsoPropyl ThioGalactoside, which induces
the synthesis of β galactosidase enzyme like lactase) and X-Gal (substrate
for β galactosidase). However, non-recombinants with intact lac-Z gene
produce blue colonies (Refer Figure 6.2).

Fig. 6.2 Genetic Map of pUC

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Bacteriophages as Cloning Vectors Cloning Vectors

Bacteriophages are in reality viruses that have the ability to infect bacterial cells
and they are widely used for cloning purposes due to their small size.
The DNA of a bacteriophage is linear shaped and it gives rise to two segments NOTES
after a single cleavage. The segments can be combined together via a foreign DNA
resulting in the formation of chimeric phages. These phages can be isolated and
inserted to infect bacteria. Progeny particles can be collected after the Lytic Cycle.
There are many phages used as cloning vectors:
6.2.2 Phage λ
It is frequently used bacteriophage for cloning purposes. Phage λ virion has a
structure which consists of its head which contains the genome and the tail fulfils
the function of attacking the host, Escherichia coli (Refer Figure 6.3).

Fig. 6.3 Structure of Phage λ and Measurement of Different Parts of a Lambda Phage

The genome of a wild type virion has a 50 kb dsDNA enclosed within a

protein coat. During infection of host the protein coat gets removed and the DNA
is transferred into the host cell.
The terminals of Phage λ consist of overhanging 5´ends that have
complementary base segments are 12 nucleotides in length. The overhanging sticky
5´ends undergo base pairing and are more sticky and cohesive than the restriction
nuclease created ends.
Due to this sticky nature this sequence is known as COS Sequence. After
entering the bacteria the COS sequences undergo base pairing and they block the
splits by using ligases. This leads to production of a ds circular DNA which takes
part in alternative fates or cycles (Refer Figure 6.4).

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Cloning Vectors

NOTES

Fig. 6.4 Chromosomal Map of Phage λ DNA Showing COS Sequence

Lytic Cycle
In this pathway the DNA undergoes replication in both directions leading to
formation of rolling circle model and hence, many linear multimers of the phage λ
are produced. These multimers are cut at the sites of cos sequences to form unit
segments of phage λ genomes. Some of these cause lysis of cell wall of host
bacterial cell and this helps in escaping the virion thus, allowing it to infect another
new host cell (Refer Figure 6.5).

Fig. 6.5 The Phage λ Undergoing Replication via Lytic and Lysogenic Cycles
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Lysogenic Cycle Cloning Vectors

Phage λ consists of a gene which has a homolog present in chromosome of

Escherichia coli. This gene is gene att. The homologues genes present in phage λ
and Escherichia coli lead to combining of phage λ DNA in the Escherichia coli NOTES
chromosome. The resulting phage λ DNA is known as PROVIRUS while the
host cell is known as LYSOGEN.
The genes which control the lysogenic cycle are present in centre of phage
λ genome. But, the fate of phage λ to undergo lytic or lysogenic cycle is under the
control of two regulatory genes which are cl gene and cro gene. They have
antagonistic in nature.
In the lytic cycle, cro gene dominates over cl gene. However, in lysogenic
cycle cl gene dominates and along with suppression of cro gene transcription it
also suppresses other genes in phage λ. Generally, the lysogenic cycle is favoured.
The genes which are involved in lysogenic cycle and not in lytic cycle are
considered non-essential in the functioning of phage λ as a cloning vector and
hence are removed leaving space in phage λ genome for the insertion of foreign
DNA.
Thus, the formed recombinant phage λ have the capability to transform the
bacterial Escherichia coli host cell at high efficacy (Refer Figure 6.6).

Fig. 6.6 Formation of Phage Particles from Phage λ

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Cloning Vectors Replacement Vectors
Phage λ genome has certain genes at central segment which are important for
lysogenic pathway but not for lytic pathway. Hence, this segment may be removed
NOTES for the insertion of foreign DNA (up to 23 kb in length) in these vectors. Generally,
these vectors are used for the formation of genomic DNA libraries.
For example, there are 2 vectors named EMBL3 and EMBL4 which help
in replacement of 44 kb long central segment from foreign desirable DNA. After
splitting of phage λ there are 3 segments/fragments that are formed, left and right
arms along with the replaceable non essential central segment known as
STUFFER.
Insertion Vectors

Vectors which are used for the synthesis of cDNA libraries are called insertion
vectors. They can be formed by modifying phage λ genome to allow insertional
cloning in gene cl.
For example, the 43 kb long ds DNA, λ gt10 helps in cloning of 7 kb
long DNA fragments. After insertion of desired DNA, the cl gene gets inactivated,
thus, generating a cl-phage. The non-recombinant λ gt10 forms cloudy plaques
in host cells and is called cl+. On the other hand, recombinant λgt10 forms clear
plaques in host cells of Escherichia coli and is called cl–. This permits the
screening of recombinant phages. Furthermore, in the bacterial strain containing
high frequency lysogeny mutation or hflA150 mutation, cl+ forms lysogens and
does not take place in lysis for formation of plaques. Thus, only cl– will form
plaques. This implies that recombinant type λ gt10 plaques can be easily opted
out.
6.2.3 Cosmid Vectors
They are synthesised by insertion of COS sequence of a particular phage into a
plasmid vector. The COS sequence helps the DNA in getting packed inside the λ
particle.
Similar to plasmids, cosmid vectors remain in the bacterial cells and do not
have genes required for lysis. Cosmids have many advantages of being used as
vectors as they have the ability of producing genomic libraries containing106 -
107 clones from 1pg of inserted DNA. But the disadvantage of cosmid vector is
that it is not able to accept lengths of DNA greater than 40-50 kbp (Refer Figure
6.7).

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 Cloning Vectors

NOTES

Fig. 6.7 Cloning in a Cosmid Vector

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Cloning Vectors 6.2.4 Phagemids
Phagemids are synthesised artificially by the combination of characteristics of phages
and plasmids, similar to cosmids. The features of phagemids are:
NOTES • They have a site for multiple cloning.
• They have a lac promoter which can be induced.
• They have a phage and plasmid derived origin of replication.
For example, phagemid pBluescript IIKS is synthesized from the plasmid
pUC19 and has length of 2961 bp. In this name KS depicts the orientation of
poly-linker such that transcription of the gene par Z begins at the restriction site
for Kpnl and Sacl. This artificially synthesized vector consists of several cloning
sites placed at the side of T3 and T7 promoters and also an inducible lac, named
lac 1which is present at the upstream of region called lac Z. The region lac Z
complements the Escherichia coli bacterium. This region uses the criteria of
formation of white colonies against blue colonies produced when foreign DNA is
not inserted and using this criterion it helps in selection of chimeric vector. This
vector consists of (Refer Figure 6.8):
• The fl (+) and fl (-) ORI which are derived from filamentous phage in order
to recover sense (+) and antisense strands (-) strands of gene named lac Z
in conditions of co-infection of host cell by helper phage.
• ColE1 ORI which is derived from plasmid in the absence of helper phage.
• Gene required for resistance to Ampicillin for the purpose of antibiotic
selection of chimeric vectors.

Fig. 6.8 Structure of pBluescript IIKS

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6.2.5 Vectors Required for the Cloning of Larger DNA Segments Cloning Vectors

Since human and many mammalian genomes are quite large, hence, such vectors
are required for their cloning. The vectors which are useful for expressing and
characterising large genes or gene complexes have recently developed (Refer Table NOTES
6.1).
Table 6.1 Maximum DNA Insert Possible with Different Cloning Vectors

Bacterial Artificial Chromosome Vectors

The vectors which are utilized for the cloning of bacterial DNA have very high
number of replicons. These, thus, produce great number of DNA clones which
depends on the replication efficacy of its replicon.
These vectors however have a disadvantage. In these vectors, the inserts
are structurally not stable. This results in rearrangement or deletion of some part of
the cloned DNA. In Eukaryotic inserts this phenomenon is very common because
in them the repetitive sequences occur frequently. Hence, the process of cloning
and maintaining a large DNA in bacteria is quite strenuous.
This limitation can be overcome by recently developed vectors which consist
of low copy number of replicons. An example of such vector is derived from
Escherichia coli called F-factor or fertility plasmid. The F-factor has 2 genes
namely par A and par B which have the function of maintaining the copy number of
F-factor at 1-2 per cell.
This vector has the ability to accept large fragment of foreign DNA like
more than 300 kb and the yielded recombinant DNA can be carried to bacterial
cells via electroporation. Electroporation is a process in which the cells are kept in
high voltage which relaxes the permeability of their plasma membrane. But, the
recombinant DNA obtained from the host cells is recovered in low yields by using
such vectors.

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Cloning Vectors Bacteriophage P1 and P1-Derived Artificial Chromosome Vectors
(PAC Vectors)
There are certain phages which possess large genomes and thus they can fit in
NOTES larger fragments of DNA. An example of such bacteriophage is PI which can
accommodate 110-115 kb long linear DNA in its protein coat. The P1 phage
components are used in the preparation of P1 cloning vectors which reside in a
circular plasmid.
The plasmid vector is severed to yield 2 vector arms which are ligated to
foreign DNA (up to 100 kb long) and then packaged into the protein coat in vitro.
The recombinant PI phage obtained adsorbs to a particular host after which the
PI DNA enters into the cell and gets amplified. Another bacteriophage T which
was packaged in P1 vectors in vitro helped in recovering inserts which were sized
up to 122 kb. Also, the features of P1 phage and F-factor have been merged to
yield PAC cloning system.
6.2.6 Yeast Artificial Chromosomes (YAC Vectors)
YAC vectors have the ability to clone very large fragments of DNA and are thus,
more useful for cloning in bacterial cells. These vectors have the following features:
• There is TEL (Yeast Telomere) at each of their ends.
• They contain a Yeast Centromere Sequence (CEN) which allows controlled
segregation during the process of mitosis.
• Each arm has a selectable marker which is used for detecting YAC in yeast.
• An ORI named ARS (Autonomously Replicating Sequence) permits the
vector to carry out replication in yeast cell.
• In order to insert foreign DNA, restriction sites which are unique to YAC
can be used.
In the cloning experiments, one of the restriction enzyme cuts at multiple cloning
sites and the other breaks between 2 TELS in circular YAC. Thus, left and right
arms are developed and a DNA of high molecular weight can be ligated to both
arms.
The external cell wall of the yeast cells is requires to be removed so as to
transfer the resulting YAC. As a result, the yeast spheroplasts that are obtained
can receive exogenous fragments but they are not stable osmotically and are required
to be embedded in agar.
Thus, the transformation efficacy as well as yielded clone DNA is very low,
but the ability of YAC vector to clone large fragments of DNA which are up to
2Mb make them a useful tool in generation of physical maps of huge genomes like
that of human genome.
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6.2.7 Vectors for the Preparation of ssDNA Cloning Vectors

The ssDNA are utilized as templates for the conventional sequencing of DNA and
they can be synthesized from PCR-based processes. These can also be prepared
from vectors which are based on some phages in which genomes bear ssDNA at NOTES
certain stages in the life cycle. Examples include the following.
M13 Bacteriophage: It is filamentous and has ss circular DNA (6.4 kb in
length) which is in close relation with other DNA sequences in the genome. This
genome is packaged inside a protein coat resulting in the filamentous form of this
bacteriophage. When the phage gets adsorbed to the host cell of Escherichia
coli, its ss form of genome gets converted into the replicative form which is the
double stranded form. Later, one of the genome products of the phage causes the
synthesis of ssDNA. These ssDNA move towards the cell membrane and gets
packaged in a protein coat. Thus, several phage particles are forced out from
Escherichia coli cell without lysis.
M13 Vectors: The production of M13 vectors and synthesis ds recombinant
DNA circles depends on the ds replicative forms which has multiple cloning sites.
M13 vectors are transferred into the host cell. Later on, after infecting the host
Escherichia coli cell these phage particles are harvested and got rid of their protein
coat which causes the release of ss recombinant DNA which can be used as
template in the process of DNA sequencing.
Additionally, phagemid vectors can also be used for the function of M13
vectors. However, in such conditions the host cells of Escherichia coli are super
infected with helper phage which is important for providing coat protein. The
resulting phage particles are a combination of helper phage and recombinant
phagemids and can be directly subjected to DNA sequencing. Some of the common
phagemid vectors are PEMBL plasmids and also pBluescript family.
Expression Vectors
In addition to amplification and propagation of cloned DNA sometimes gene
expression is also necessary. Hence, in these conditions particular expression signals
are required to be transmitted by cloning system. Thus, several expression cloning
vectors are utilised in variety of host cell systems like bacterial cells or mammalian
cells.
An expression cloning vector is the one which contains regulatory sequences
that are mandatory for permitting the process of transcription and translation to
occur in cloned gene(s). It is fabricated for simply watching out the expression.
However, this process may also help to recover huge amount of expression product
which is necessary to yield large amount of particular protein that is pharmaceutically
vital.
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Cloning Vectors These are derivatives of plasmid cloning vectors that are used inside the
host cell. However, there are some moderations like addition of a promoter which
are specific to the host so as to permit transcription of the cloned gene; transcription
signal as well if it is suitable for the host.
NOTES
In prokaryotes and eukaryotes various mechanisms are used by translation
system to recognize the start codon. For instance, in Escherichia coli presence
of Shine-Dalgarno sequence upstream of start codon is necessary for the expression
of a specific Eukaryotic gene.
The transcription of a specific gene is depends on functioning of its promoter.
Thus, a gene which has to be investigated and is easily assayable is usually merged
behind its promoter so as to know the quantity of reporter gene produced under
specific conditions. This data can be utilized to study the gene as well as the
expression of its promoter in a more sophisticated manner.
An important aspect of gene expression is codon bias. All amino acids
(except tryptophan and methionine ) are coded by 2 or more than 2 codons but
not all of those codons which encode for only 1 amino acid are utilized in an equal
amount. For example, there are 4 codons which encode Analine and those codons
are GCA, GCC, GCG and GCT.
However, in humans GCG is used four times less than GCC for encoding
alanine. This is known as CODON BIAS where there biasing for one or more
than one codons against other codons. Hence, for the required protein to be
produced at highest level, certain modifications in the coding sequence of cloning
gene need to be done so as to make sure that correct codon bias exists. This is
known as codon optimization. There is an insecticidal protein named ‘cry’ which
when obtained from bacteria had very low expression level due to codon bias
which favoured translation in bacteria. This protein, however, was expressed in
plant system after codon optimisation and conferred resistance against pests.
Host Engineering
Sometimes the genetic makeup of host greatly affects the expression of cloned
gene. The protease enzyme in host may degenerate the cloned protein and this
may cause recombination of introduced heterogeneous gene. Thus, to tackle such
problems certain modifications are done in the host which is known as host
engineering.
For instance in Escherichia coli, for the expression of cloned genes protease
deficient ion mutant is used. So to reduce the occurrence of DNA recombination,
RecA mutant is utilized which presents less recombination of cloned DNA resulting
in enhanced expression of cloned genes.
Expression in Bacteria
Prokaryotic cells like bacterial cells have promoters such as trp genes or lac
genes which are prone to get induced in existence of chemicals, such as
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IsoPropylThioGalactoside (IPTG). Present vectors used in bacteria have artificial Cloning Vectors

promoters, such as Tac obtained from Trp, Lac and PL. These amalgamate high
expression characteristics of various promoters are having the ability to provide
greater expression values in comparison to single native promoter. pET vectors
use phage T7 promoters to control production of cloned gene products. NOTES
Bacteria do not have the ability of processing introns and thus, cloned gene
that is to be expressed is free from introns. This type of a gene is now cloned
downstream of the specific promoter. Usually, the cloned gene is manifested as a
fusion protein having maltose- binding protein or glutathione-s-transferase. This
aids in affinity purification of fusion protein by moving bacterial extract via a column
consisting of glutathione agarose or amylase resin beads. The bound fusion protein
is extracted from column and a protease named Factor Xa cleaves it between the
Amino Acids– Arginine and Valine.
The vectors orderly consist of:
• Sequences of N-Terminal Amino Acids of Bacterial Gene.
• Arginine and Valine Specific Codons which acts as Sites of Cleavage by
Factor Xa.
• Restriction Sites for introducing the Cloned Gene that is needed to be
expressed in the Vector.
Expression in Yeast

Eukaryotic proteins require certain modifications in their functional activity after

translation which does not happen in the bacterial host. So, the Eukaryotic
expression system is more advantageous than Prokaryotic vectors. Hence, for
such purpose various species of yeast have been utilized, such as Sacchromyces
cervisiae, Pichia pastoris, and many more.
Usually yeast expression vectors are synthesized by combining a small DNA
fragment derived from yeast named as 2 µm fragment with a bacterial plasmid.
The small DNA fragment derived from yeast acts as the origin of replication and
hence, provides the needed properties of replication. A cloned yeast promoter
controls the expression of the cloned gene.
Further, the expression system consists of a particular yeast gene such as
leu which aids the growth of transformed leu yeast in a leucine lacking growth
medium. This also serves in selection of transformed yeast cells. Plasmids are also
joined into the yeast chromosome via homologous recombination which makes
sure that the cloned gene is maintained in a stable manner. The easy growth in any
media and economically friendly behaviour of yeasts make them perfect for gaining
the proteins of interest.
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Cloning Vectors Expression in Mammalian Cells
There are various mammalian vectors which are used for the expression of cloned
genes in mammalian cells. These are:
NOTES • Papoviruses, such as SV40 and adenoviruses are DNA viruses which can
be used for the expression of cloned genes in mammalian cells. However,
they are not good for the expression of large genes because they low capacity
for foreign DNA.
• For the expression in mammalian cells even retroviruses like murine
retrovirus, are also used and they have the ability to attack great species of
cell type and also have great capacity for foreign DNA. They have the
disadvantage that they are ssRNA and without getting integrated they cannot
undergo replication. Hence, retroviruses need replicating cells for expression.
• The third example of vectors is adeno-associated virus which is non-
pathogenic and can also overcome the disadvantages regarding retroviruses.
• Another one is Herpes virus which has a large size and very needful in non-
integrating. It can also be utilized for the expression of larger genes.
• The pathogen activity of these viruses must be removed when using them as
vectors such that only those genes which are associated with replication or
have high copy number remain. The vectors also require a selection marker
like neomycin resistance gene which gives resistance to G418 (a cytotoxic
compound).
Special Vectors
Apart from the above mentioned vectors there are three more vectors:
Transposons Vectors
Drosophila has an element named the P element which is an essential transposon
and contains 31 base pairs terminal repeats which surround a 3 kbp region for
protein coding. This protein coding region codes for the protein transposase and
transposition repressor.
But, there are also various deleted P elements which do not contain
transposase or other such genes. In such transposons, the required gene is inserted
which gives rise to recombinant P elements. The recombinant P elements are further
microinjected in fertilized eggs with transposase containing normal P element.
Hence, the gene of interest is able to get transposed onto embryonic chromosome.
Plasmid Shuttle as Vector
Shuttle vectors or cloning vectors are those vectors which can be used to transform
mammalian as well as animal cells, plant cells, or yeast cells in cultures. For instance,
certain shuttle vectors can be changed into and replicated in Escherichia coli and
can also be modified into yeast.
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Retriever Vectors Cloning Vectors

Retriever vectors are a set of vectors which are utilized to retrieve certain genes
from normal chromosome via recombination. Such vectors have the ability to
multiply in both Escherichia coli and yeast cells. In such vectors, the required
NOTES
gene can be deleted by enzyme so as to yield gapped vector consisting of
encompassing sequences.
Retriever vectors can be very handy in isolating genes from yeast for further
studies because via recombination they have the ability to retrieve lost sequences
from host.
6.2.8 Viral Vector – SV 40 and Adenovirus
Viral vectors are tools commonly used by molecular biologists to deliver genetic
material into cells. This process can be performed inside a living organism (in vivo)
or in cell culture (in vitro). Viruses have evolved specialized molecular mechanisms
to efficiently transport their genomes inside the cells they infect. Delivery of genes,
or other genetic material, by a vector is termed transduction and the infected cells
are described as transduced. Molecular biologists first harnessed this machinery
in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the
bacteriophage l to infect monkey kidney cells maintained in culture.
In addition to their use in molecular biology research, viral vectors are used
for gene therapy and the development of vaccines.
Key Properties of a Viral Vector
Viral vectors are tailored to their specific applications but generally share a few
key properties.
Safety: Although viral vectors are occasionally created from pathogenic
viruses, they are modified in such a way as to minimize the risk of handling them.
This usually involves the deletion of a part of the viral genome critical for viral
replication. Such a virus can efficiently infect cells but, once the infection has taken
place, requires a helper virus to provide the missing proteins for production of
new virions.
Low Toxicity: The viral vector should have a minimal effect on the physiology
of the cell it infects.
Stability: Some viruses are genetically unstable and can rapidly rearrange
their genomes. This is detrimental to predictability and reproducibility of the work
conducted using a viral vector and is avoided in their design.
Cell Type Specificity: Most viral vectors are engineered to infect as wide
a range of cell types as possible. However, sometimes the opposite is preferred.
The viral receptor can be modified to target the virus to a specific kind of cell.
Viruses modified in this manner are said to be pseudotyped.
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Cloning Vectors Identification: Viral vectors are often given certain genes that help identify
which cells took up the viral genes. These genes are called markers. A common
marker is resistance to a certain antibiotic. The cells can then be isolated easily, as
those that have not taken up the viral vector genes do not have antibiotic resistance,
NOTES and so cannot grow in a culture with the relevant antibiotic present.
Adenoviruses
Adenoviruses are a group of viruses that typically cause respiratory illnesses, such
as a common cold, conjunctivitis (an infection in the eye that is sometimes called
pink eye), croup, bronchitis, or pneumonia. In children, adenoviruses usually cause
infections in the respiratory tract and intestinal tract. Consider the following facts
about adenoviruses:
• Infection in children may occur at any age.
• Adenoviral respiratory infections are most common in the late winter, spring,
and early summer. Adenoviruses can occur anytime throughout the year.
• Digestive tract infections are more common in children under the age of 5.
• Most children have had one form of the infection by age 10.
The following are the most common ways adenoviruses are transmitted:
Respiratory Infections: Respiratory infections occur by coming in contact with
infectious material from another individual or inanimate object. The secretions
from the respiratory tract may contain the virus. The virus can also survive for
many hours on inanimate objects, such as doorknobs, hard surfaces, and toys.
Intestinal Tract Infections: Transmission of the digestive strain of the virus usually
occurs by fecal-oral contact. Usually this occurs from poor hand washing or from
ingestion of contaminated food or water.
SV40 Virus
SV40 is an abbreviation for Simian Vacuolating Virus 40 or Simian Virus 40, a
polyomavirus that is found in both monkeys and humans. It was named for the
effect it produced on infected green monkey cells, which developed an unusual
number of vacuoles. Like other polyomaviruses, SV40 is a DNA virus that has
the potential to cause tumors in animals, but most often persists as a latent infection.
SV40 has been widely studied as a model eukaryotic virus, leading to many early
discoveries in eukaryotic DNA replication and transcription.
SV40 consists of an un-enveloped icosahedral virion with a closed circular
dsDNA genome of 5.2 kb. The virion adheres to cell surface receptors of MHC
class I by the virion glycoprotein VP1. Penetration into the cell is through a caveolin
vesicle. Inside the cell nucleus, the cellular RNA polymerase II acts to promote
early gene expression. This results in an mRNA that is spliced into two segments.
The small and large T antigens result from this. The large T antigen has two functions
- 5% goes to the plasma cell membrane and 95% returns to the nucleus. Once in
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the nucleus the large T antigen binds three Viral DNA Sites, I, II and III. Binding Cloning Vectors

of Sites I and II auto-regulates early RNA synthesis. Binding to Site II takes place
in each cell cycle. Binding Site I initiates DNA replication at the origin of replication.
Early transcription gives two spliced RNAs that are both 19s. Late transcription
gives both a longer 16s, which synthesizes the major viral capsid protein VP1; NOTES
and the smaller 19s, which gives VP2 and VP3 through leaky scanning. All of the
proteins, besides the 5% of large T, return to the nucleus because assembly of the
viral particle happens there. Eventual release of the viral particles is cytolytic and
results in cell death.

Check Your Progress

1. Give the salient features of plasmids.
2. What are the features that plasmid should have so that it acts as a cloning
vector?
3. Write a short note on phage λ.
4. Explain lysogenic cycle.
5. List the features of YAC vectors.
6. What is host engineering? Explain with the help of example.
7. Explain what viral vectors are? What are adenoviruses?
8. What is SV40 virus? What is its significance?

6.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. Salient features of plasmids are as follows:

• Plasmids may be single copy plasmids as they are present as one plasmid
DNA per bacterial cell.
• Plasmids may be multicopy plasmids as they are maintained as 10-12
genomes per host cell.
• Plasmids are also capable of increasing their copy number in the host
cell up to 100 copies under relaxed replication control.
2. To act as cloning vectors, all plasmids should possess the following properties:
• They should be easily isolated from their host cells.
• They should contain single restriction site for at least one or more
restriction enzymes.
• Inserting a desirable piece of a linear foreign DNA at one end of these
sites should not modify the replication properties of plasmid.

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Cloning Vectors • Plasmids can be easily reintroduced back into the host bacterial cells.
• Bacterial host cells carrying the plasmid with or without foreign insert
should be effortlessly selected or identified.
NOTES 3. Phage λ is frequently used bacteriophage for cloning purposes. Phage λ
virion has a structure which consists of its head which contains the genome
and the tail fulfils the function of attacking the host, Escherichia coli. The
genome of a wild type virion has a 50 kb dsDNA enclosed within a protein
coat. During infection of host the protein coat gets removed and the DNA
is transferred into the host cell. The terminals of Phage λ consist of
overhanging 5´ends that have complementary base segments are 12
nucleotides in length. The overhanging sticky 5´ends undergo base pairing
and are more sticky and cohesive than the restriction nuclease created ends.
4. Phage λ consists of a gene which has a homolog present in chromosome of
Escherichia coli. This gene is gene att. The homologues genes present in
phage λ and Escherichia coli lead to combining of phage λ DNA in the
Escherichia coli chromosome. The resulting phage λ DNA is known as
PROVIRUS while the host cell is known as LYSOGEN. The genes which
control the lysogenic cycle are present in centre of phage λ genome. But,
the fate of phage λ to undergo lytic or lysogenic cycle is under the control
of two regulatory genes which are cl gene and cro gene. They have
antagonistic in nature.
5. Yeast Artificial Chromosomes (YAC) vectors have the ability to clone very
large fragments of DNA and are thus, more useful for cloning in bacterial
cells. These vectors have the following features:
• There is TEL (Yeast Telomere) at each of their ends.
• They contain a Yeast Centromere Sequence (CEN) which allows
controlled segregation during the process of mitosis.
• Each arm has a selectable marker which is used for detecting YAC in
yeast.
• An ORI named ARS (Autonomously Replicating Sequence) permits
the vector to carry out replication in yeast cell.
• In order to insert foreign DNA, restriction sites which are unique to
YAC can be used.
6. Sometimes the genetic makeup of host greatly affects the expression of
cloned gene. The protease enzyme in host may degenerate the cloned protein
and this may cause recombination of introduced heterogeneous gene. Thus,
to tackle such problems certain modifications are done in the host which is
known as host engineering. For instance in Escherichia coli, for the
expression of cloned genes protease deficient ion mutant is used. So to
reduce the occurrence of DNA recombination, recA mutant is utilized which
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 presents less recombination of cloned DNA resulting in enhanced expression Cloning Vectors

of cloned genes.
7. Viral vectors are tools commonly used by molecular biologists to deliver
genetic material into cells. This process can be performed inside a living NOTES
organism (in vivo) or in cell culture (in vitro).
Adenoviruses are a group of viruses that typically cause respiratory illnesses,
such as a common cold, conjunctivitis (an infection in the eye that is
sometimes called pink eye), croup, bronchitis, or pneumonia. In children,
adenoviruses usually cause infections in the respiratory tract and intestinal
tract.
8. SV40 is an abbreviation for Simian Vacuolating Virus 40 or Simian Virus
40, a polyomavirus that is found in both monkeys and humans. It was named
for the effect it produced on infected green monkey cells, which developed
an unusual number of vacuoles. Like other polyomaviruses, SV40 is a DNA
virus that has the potential to cause tumors in animals, but most often persists
as a latent infection. SV40 has been widely studied as a model eukaryotic
virus, leading to many early discoveries in eukaryotic DNA replication and
transcription.

6.4 SUMMARY

• A cloning vector is a small piece of DNA that can be stably maintained in an

organism, and into which a foreign DNA fragment can be inserted for cloning
purposes.
• The cloning vector may be DNA taken from a virus, the cell of a higher
organism, or it may be the plasmid of a bacterium.
• After a DNA fragment has been cloned into a cloning vector, it may be
further subcloned into another vector designed for more specific use.
• There are many types of cloning vectors, but the most commonly used ones
are genetically engineered plasmids.
• Cloning is generally first performed using Escherichia coli, and cloning
vectors in Escherichia coli include plasmids, bacteriophages (such as,
Phage l), cosmids, and Bacterial Artificial Chromosomes (BACs).
• Some DNA, however, cannot be stably maintained in Escherichia coli, for
example very large DNA fragments, and other organisms, such as yeast
may be used. Cloning vectors in yeast include Yeast Artificial Chromosomes
(YACs).
• Consequently, the cloning vector contains features that permit for the easy
insertion or removal of a DNA fragment to or from the vector.
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Cloning Vectors • Plasmids are extra chromosomal, double-stranded DNA molecules that
replicate autonomously within bacterial cells.
• Most of the plasmids are circular in structure but linear plasmids have been
NOTES found in some bacteria like Borrelia burgdorferi and Streptomyces.
• The size of the naturally occurring plasmids ranges from a few thousand base
pairs to more than 100 kilo-bases (kb).
• Replication of plasmids depends upon the same set of enzymes that are
responsible for the replication of chromosome of the host cells.
• Plasmids generally contain genes that help the host cells. For instance, a
bacterial cell containing plasmids that encodes antibiotic inactivating enzymes
can replicate in an environment containing the antibiotic while other bacterium
lacking the drug-resistant plasmid failed to thrive.
• Plasmids are the first vector that were developed for gene cloning experiments
and till date are widely used due to their versatile nature.
• Most plasmids commonly used as cloning vector in molecular biology,
replicate in the bacterium Escherichia coli.
• Length is shortened to easily work with plasmids; many such plasmids used
as cloning vectors are only 3 kb in length, much shorter than their original
lengths.
• Most plasmid used as cloning vectors contain little more than the necessary
nucleotide sequences essential for their use in DNA cloning: a drug-resistance
gene, a replication origin and a region in which exogenous foreign DNA
fragment can be inserted.
• Plasmids to be used as cloning vectors are also customized to increase their
copy number in host bacterial cells by the process of amplification.
• pBR group of plasmids is the most widely used cloning vectors named after
the discoverer of these plasmids namely Bolivar and Rodriguez.
• Cloning of foreign DNA in pBR322 with the aid of any one of these
restriction enzymes will lead to insertional inactivation of either the TetR or
AmpR markers.
• Bacteriophages are in reality viruses that have the ability to infect bacterial
cells and they are widely used for cloning purposes due to their small size.
• The DNA of a bacteriophage is linear shaped and it gives rise to two
segments after a single cleavage.
• The segments can be combined together via a foreign DNA resulting in the
formation of chimeric phages. These phages can be isolated and inserted to
infect the bacteria.
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• The genome of a wild type virion has a 50 kb dsDNA enclosed within a Cloning Vectors

protein coat. During infection of host the protein coat gets removed and the
DNA is transferred into the host cell.
• The terminals of Phage λ consist of overhanging 5´ ends that have
NOTES
complementary base segments are 12 nucleotides in length. The overhanging
sticky 5´ ends undergo base pairing and are more sticky and cohesive than
the restriction nuclease created ends.
• Due to this sticky nature this sequence is known as COS Sequence. After
entering the bacteria the COS sequences undergo base pairing and they
block the splits by using ligases.
• In this pathway the DNA undergoes replication in both directions leading to
formation of rolling circle model and hence, many linear multimers of the
phage λ are produced. These multimers are cut at the sites of COS sequences
to form unit segments of phage λ genomes.
• Phage λ consists of a gene which has a homolog present in chromosome of
Escherichia coli.
• The homologues genes present in phage λ and Escherichia coli lead to
combining of phage λ DNA in the Escherichia coli chromosome. The
resulting phage λ DNA is known as PROVIRUS while the host cell is
known as LYSOGEN.
• The genes which control the lysogenic cycle are present in centre of
phage λ genome. But, the fate of phage λ to undergo lytic or lysogenic
cycle is under the control of two regulatory genes which are cl gene and cro
gene. They have antagonistic in nature.
• Phage λ genome has certain genes at central segment which are important
for lysogenic pathway but not for lytic pathway.
• Vectors which are used for the synthesis of cDNA libraries are called insertion
vectors. They can be formed by modifying phage λ genome to allow
insertional cloning in gene cl.
• The vectors which are utilized for the cloning of bacterial DNA have very
high number of replicons. These, thus, produce great number of DNA clones
which depends on the replication efficacy of its replicon.
• These vectors however have a disadvantage. In these vectors, the inserts
are structurally not stable. This results in rearrangement or deletion of some
part of the cloned DNA.
• In Eukaryotic inserts this phenomenon is very common because in them the
repetitive sequences occur frequently.
• The plasmid vector is severed to yield 2 vector arms which are ligated to
foreign DNA (up to 100 kb long) and then packaged into the protein coat
in vitro.
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Cloning Vectors • The recombinant PI phage obtained adsorbs to a particular host after which
the PI DNA enters into the cell and gets amplified.
• The production of M13 vectors and synthesis ds recombinant DNA circles
NOTES depends on the ds replicative forms which has multiple cloning sites. M13
vectors are transferred into the host cell.
• In Prokaryotes and Eukaryotes various mechanisms are used by translation
system to recognize the start codon.
• Viral vectors are tools commonly used by molecular biologists to deliver
genetic material into cells. This process can be performed inside a living
organism (in vivo) or in cell culture (in vitro).
• Delivery of genes, or other genetic material, by a vector is termed
transduction and the infected cells are described as transduced. Molecular
biologists first harnessed this machinery in the 1970s.
• Adenoviruses are a group of viruses that typically cause respiratory illnesses,
such as a common cold, conjunctivitis (an infection in the eye that is
sometimes called pink eye), croup, bronchitis, or pneumonia. In children,
adenoviruses usually cause infections in the respiratory tract and intestinal
tract.
• SV40 is an abbreviation for Simian Vacuolating Virus 40 or Simian Virus
40, a polyomavirus that is found in both monkeys and humans. It was named
for the effect it produced on infected green monkey cells, which developed
an unusual number of vacuoles.
• Like other polyomaviruses, SV40 is a DNA virus that has the potential to
cause tumors in animals, but most often persists as a latent infection.
• SV40 has been widely studied as a model Eukaryotic virus, leading to
many early discoveries in Eukaryotic DNA replication and transcription.

6.5 KEY WORDS

• Cloning vector: A cloning vector is DNA fragment derived from a virus or

plasmid of bacterium or cell of higher organism which can be inserted into a
foreign DNA for the purpose of cloning.
• Plasmids: Plasmids are extra chromosomal, double-stranded DNA
molecules that replicate autonomously within bacterial cells.
• Retriever vectors: Retriever vectors are a set of vectors which are utilized
to retrieve certain genes from normal chromosome via recombination.
• Insertion vectors: Vectors which are used for the synthesis of cDNA
libraries are called insertion vectors.
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 • Viral vectors: These are tools commonly used by molecular biologists to Cloning Vectors

deliver genetic material into cells, this process can be performed inside a
living organism (in vivo) or in cell culture (in vitro).
• Adenoviruses: These are a group of viruses that typically cause respiratory
NOTES
illnesses, such as a common cold, conjunctivitis (an infection in the eye that
is sometimes called pink eye), croup, bronchitis, or pneumonia.
• SV40: It is an abbreviation for Simian Vacuolating Virus 40 or Simian Virus
40, a polyomavirus that is found in both monkeys and humans.

6.6 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. Write short note on bacteriophages as cloning vectors.
2. What are cosmid vectors?
3. Explain phasmids as vectors.
4. Distinguish between Plasmid Artificial Chromosome (PAC) and Yeast
Artificial Chromosome (YAC).
5. What are M13 bacteriophages and M13 vectors?
6. What are viral vectors?
Long Answer Questions
1. Define cloning vectors. List the characteristics of a vector.
2. Discuss in detail the pBR322 and pUC vectors.
3. Differentiate between plasmid, bacteriophages and phasmids vectors.
4. Elaborate a note on vectors for cloning large DNA fragment.
5. Briefly discuss about the Plasmid Artificial Chromosome (PAC) and Yeast
Artificial Chromosome (YAC).
6. What are expression vectors? Discuss with examples.
7. Discuss about the viral vectors – SV 40 and adenovirus.

6.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
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Cloning Vectors Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
NOTES Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 Cloning of Human
BLOCK - III Insulin
GENE CLONING

NOTES
UNIT 7 CLONING OF HUMAN
INSULIN
Structure
7.0 Introduction
7.1 Objectives
7.2 Cloning of Human Insulin
7.2.1 Methodology
7.2.2 Synthesis of Interferon in Escherichia coli
7.2.3 Development of Recombinant Vaccines
7.3 Answers to Check Your Progress Questions
7.4 Summary
7.5 Key Words
7.6 Self Assessment Questions and Exercises
7.7 Further Readings

7.0 INTRODUCTION

Gene cloning is the process in which a gene of interest is located and copied out of
DNA extracted from an organism. When DNA is extracted from an organism, all
of its genes are extracted at one time. This DNA, which contains thousands of
different genes. The genetic engineer must find the one specific gene that encodes
the specific protein of interest.
The production of exact copies of a particular gene or DNA sequence using
genetic engineering techniques. The DNA containing the target gene(s) is split into
fragments using restriction enzymes. These fragments are then inserted into
cloning vectors, such as bacterial plasmids or bacteriophages, which transfer the
recombinant DNA to suitable host cells, such as the bacterium Escherichia coli.
Alternatively, complementary DNA is inserted into the vectors, or ‘naked’ DNA
fragments can be taken up directly by a host bacterium from its medium.
Inside the host cell the recombinant DNA undergoes replication; thus, a
bacterial host will give rise to a colony of cells containing the cloned target gene.
Various screening methods may be used to identify such colonies, enabling them
to be selected and cultured. Gene cloning facilitates DNA sequencing; it also enables
large quantities of a desired protein product to be produced, human insulin, for
example, is now produced by bacteria containing the cloned insulin gene.
In this unit, you will study about concept of human insulin, interferon synthesis
in Escherichia coli, recombinant vaccine development, HBs Ag in yeast in detail.
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Cloning of Human
Insulin 7.1 OBJECTIVES

After going through this unit, you will be able to:

NOTES • Understand the concept of human insulin
• Obtain a brief idea of recombinant protein and hormone synthesis
• Discuss the concept of recombinant vaccines

7.2 CLONING OF HUMAN INSULIN

Gene cloning is an advantageous process which leads to production of recombinant

proteins, hormones and various antigens required for vaccination. The major
purpose of gene cloning is identification of the protein isolated through expression
vector, which yields good amount necessary for crystallography and
characterization studies. The main clinical application of gene cloning for important
hormones and antigens are associated with medication and vaccination. The
implication of various plasmids obtained from Escherichia coli facilitates the
process of gene cloning. The use of specific set of restriction enzymes and ligases
is important to integrate the Gene Of Interest (GOI) within the plasmid vector
designed for cloning. Furthermore, the use of application vector is crucial to obtain
sufficient amount of the protein required for clinical use. The efficiency in the
production of recombinant proteins appears to be a beneficial consequence of the
completion of human genome project. The rDNA expression products have
popularised the use of modern medicines associated with hormones of therapeutic
interest, haemopoietic growth factors, blood coagulation products, thrombolytic
agents, anticoagulants, interferons, interleukins and therapeutic enzymes. Production
of human insulin through recombinant DNA methodology is an example of first
drug or recombinant hormone used for commercial purpose. Genentech in the
year 1978 originally developed the rDNA mediated production of human insulin
in a commercial scale. Henceforth, the molecule was approved by Food and
Drug Administration for therapeutic use of diabetes.
Historical Perspective
In the early years of 1970s insulin was extracted from the frozen pancreas tissues
of pigs and cattle. Insulin is a hormone made by the pancreas that allows your
body to use sugar (glucose) from carbohydrates in the food that you eat for energy
or to store glucose for future use. Insulin helps keeps your blood sugar level from
getting too high (Hyperglycemia) or too low (Hypoglycemia). This method was,
however, inconvenient and unethical. About 800 pounds of pancreas tissue yielded
only one pound of insulin. A large number of animals were required to cope with
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the high demand of this drug which is commonly used as a treatment in diabetics. Cloning of Human
Insulin
In 1978, Genentech scientist Dennis Kleid visited factories in Indiana where large
amount of cattle and pig-derived pancreas tissue was implied for extraction of
insulin. Genentech had expertise in the production of recombinant insulin by cloning
NOTES
in bacterial DNA. However, this method was not successful in yielding the drug in
sufficient amounts and hence the method was not economically viable for
commercial use. Eli Lilly was the main manufacturer of insulin in U.S. who signed
contracts with the other engineers of competing institutions. There was a need to
synthesize bioengineered insulin in laboratories. Meanwhile, Harvard and University
of California (San Francisco) had already been working with rat insulin genes to
imply rDNA technology for insulin production. The team of Genentech had only
12 members involved in the innovative research necessary to solve the needful.
The Genentech team had already popularized the production of somatostatin in
laboratory. However, the splicing method used to produce somatostatin required
to be improved before being applied to insulin. The structure of insulin is more
complex with two peptide chains present in its tertiary structure. There was a
requirement of two chain-producing DNA molecules as cloning vectors operating
in two different bacterial cells. Effectively the two chains of insulin were engineered
to be joined in the same cloning plasmid vector and transferred to a benign
Escherichia coli bacterium. The bacterium used for the transfer of cloning plasmid
yielded the two protein chains of insulin by utilizing its own protein synthesis
machinery. The two separate peptide chains obtained were joined to reconstitute
the functional unit of human pancreas insulin hormone molecule. Goeddel succeeded
in this attempt on August 21, 1978.
7.2.1 Methodology
The process of cloning of human insulin gene involved separate cloning of
polypeptide chains into the plasmid followed by the formation of recombinant
protein (Refer Figures 7.1 and 7.2).
• Enzymes: The enzymes required for GOI integration are namely T4 DNA
ligase and T4 polynucleotide kinase. Restriction endonuclease required are
ECOR1 and HindIII.
• Isolation of Cloning Plasmid: The plasmid required to clone the GOI is
pBR322 which is extracted using the following extraction medium which
contains kinase buffer, 60 mM Tris-HCl, pH 8/15 mM 2-mercaptoethanol
/10 mM MgCl2; ligase buffer, 20mM Tris-HCl, pH 7.5/10 mM dithiothreitol/
10 mM MgCl2; BamHI buffer, 20 mM Tris-HCl, pH 7.5/7 mM MgCl2/2
mM 2-mercaptoethanol; EcoRI-HindIll buffer, BamHI buffer containing 50
mM NaCl; and phosphatase buffer, 50 mM Tris-HCl, pH 8/10 mM MgCl2.
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Cloning of Human The plasmid was designed to possess β-galactosidase gene and ampicillin
Insulin
(antibiotic) resistance gene required as marker and reporter genes.
• Assembly of Insulin Gene: The genes for the two polypeptide chains of
NOTES insulin A and B are engineered by help of oligonucleotides and T4 DNA
ligase which results in the formation of specific restriction enzyme sites in
their structure. This step is necessary to clone the genes in the pBR322
plasmid. The gene fragments for A and B chain possess ECORI, BamHI
and Hind III restriction sites in their flanking regions.
• Construction of Plasmid for Expression of A and B Chain of Insulin:
The pIA1 and pIB1 plasmids are designed to produce the respective
expression vectors for chain A and chain B, respectively. The two plasmids
are digested by EcoRI and HindIII enzymes and ligated with the GOI
fragment and most of pBR322 plasmid. Restriction analysis is performed
to check the correct insertion of ampicillin and β-galactosidase gene in the
plasmid.
• Detection of Expression and Insulin Chain Assembly: The strains of
bacteria which contain the β-galactosidase-insulin A/B chain produce 30-
50% of their cellular protein as the insulin chain. The expression of proteins
can be detected by radio-immuno assay or ELISA method to confirm the
presence of polypeptide chain A and B being produced from the respective
expression vectors. The β-galactosidase-insulin hybrid chains are cleaved
by cyanogens bromide and S-sulfonated. Followed by this step the two
chains are reassembled to produce the functional conformation of protein.
The protein can be isolated and purified by reverse phase DEAE-cellulose
HPLC.

A. B.

Fig. 7.1 A. Transformed Colonies of Escherichia coli Containing the Recombinant

Plasmid DNA; B. The Map of Expression Vector pIB1 used for Cloning
B Chain of Insulin

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NOTES

Fig. 7.2 Schematic Representation of Insulin Gene Cloning and

Production of Recombinant Protein

7.2.2 Synthesis of Interferon in Escherichia coli

Interferons were discovered in the year 1957 by A. Issacs and J. Lindennmann
at the National institute of medical research in London. The human interferons
are a class of homologous proteins necessary for anti-viral, anti-proliferative and
immuno-modulatory function in the cells. The interferons belong to the cytokine
class of secretary proteins mainly involved in triggering the defense response
associated with cell to cell signaling. The name interferon has been derived from
their ability to interfere with the process of viral replication in the infected cells.
They mainly function by triggering the activity of macrophages and antigen
formation (Major Histocompatibiltiy Complex; MHC). Formation of interferons
within the cells may be manifested by fever or flu-like symptoms. The three classes
of interferon (Types I, II and III) are roughly encoded by around 20 genes
identified in animals and humans. All classes of interferons usually share common
properties in terms of inhibition of viral replication. Interferons are mostly produced
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Cloning of Human in response to bacterial and viral infection in the cells. Various external pathogen
Insulin
signaling molecules like glycoproteins, bacterial endotoxins, viral RNA may trigger
the synthesis of IFs within the cell. In the downstream cascade of IF signaling
they activate a set of STAT transcription factors which trigger immune response
NOTES
in cells. Although the IFs are capable of interfering with the replication of viruses,
there are also certain strains of viruses resistant to the activity of interferons. The
activity of the viruses results in the non-functionality of the cytokines binding to
the receptors. This results in inhibition in the formation of interferon molecules.
Various types of interferon molecules like interferon beta 1a and interferon
beta 1b are implied for the control of diseases like multiple sclerosis and auto-
immune disorders. Interferon therapy supplemented with radio-therapy appears
beneficial to the treatment of certain cases of haematological malignancy.
Recombinant IFα2 types are effective in the treatment of hepatitis and herpes
simplex viruses (Refer Figure 7.3). The first use of artificial human IF as a drug
molecule was approved by Food and Drug Administration in the year 1986. Recent
developments in the year 2001 have resulted in the production of PEGylated IFα.
The PEGylated drug form of IF has an advantage of higher retention within the
body which increases the efficacy of the drug.

Fig. 7.3 3-D Structure of a Recombinant IF (α)

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Methodology of Interferon Synthesis in Escherichia coli Cloning of Human
Insulin
The process of IF synthesis in Escherichia coli is accomplished by the construction
of a cDNA library preferably from the mRNA of human fibroblast IF. The bacterial
clone of fibroblast IF is hybridized to the cDNA probe for its detection. The NOTES
cDNA is synthesized using radioactive deoxyoligotide nucleotide primers. The
mRNA for human IF is transferred into the expression vector of Escherichia coli
to obtain good amount of IF yield. The steps involved in the process of cloning
and synthesis of IF is as follows:
• Chemical Synthesis of Oligonucleotides: The primer fragments are
synthesized on the basis of the gene sequence of the protein (IF). Based on
the information available in databases, around six set of primers are preferably
produced with 5´ and 3´ end complementation.
• Induction of Fibroblasts: Human fibroblast cell lines (preferably GM-
2504A) are grown in essential medium growth solution containing 50 mg/
ml cycloheximide. Poly (I): Poly (C) and 10 mg/ml cycloheximide. The cell
lines are incubated at 37°C for 4 hours and washed in PBS containing
0.14M NaCl, 3mM KCl, 1.5 mM KH2PO4, and 8mM Na2HPO4. Cell
line cultures are further incubated with 10 ml of a trypsin - EDTA solution
and centrifuged at 500 g for 15 min. The pellets are refrigerated.
• Extraction and Assay of Interferon mRNA: The poly-A containing
mRNA is extracted from the cell lines using standard phenol extraction
method. The mRNA can be separated and detected by oligo (dT) cellulose
chromatography or gel electrophoresis. The extract containing the poly A
interferon mRNA is intensified by using sucrose (5-10%) density gradient
centrifugation. The extracted mRNA is injected into Xenopus laevis oocytes
and incubated for 24 hours at 21°C. Followed by incubation the oocytes
are homogenised and centrifuged at 10,000 g. The activity of interferon is
detected by cytopathic effect inhibition assay using Sindbis virus human
diploid cells.
• Synthesis and Cloning of cDNA: The cDNA molecules are synthesized
with the help of mRNA of IF along with 20 mM Tris-HCl (pH 8.3), 20 mM
KCl, 8mM MgCl2' 30 mM o-mercaptoethanol, 100 1Ci of (a32P) dCTP
and 1mM dATP, dCTP, dGTP, dTTP. Primers used may preferably contain
pentamer to decamer oligonucleotide with Hind III site. Reverse transcriptase
(100 U) is added and incubated at 42°C for 30 min. The double stranded
cDNA is cleaved by endonuclease for 2 hours at 37°C in 25 mM sodium
acetate (pH 4.5), l mM ZnCl2, 0.3M NaCl. Followed by cleaving the single
strand is annealed to pBR322 plasmid and transferred to Escherichia coli
strains.
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Cloning of Human • Preparation of 32P cDNA Probe: The required amount of mRNA
Insulin
is combined with primer pools or oligo (dT) mixture containing 10mM
Tris-HCl (pH 8), 1 mM EDTA. After boiling and cooling the primer template
NOTES is mixed with 40 mM Tris-HCl (pH 8.3), 40 mM KCl, 16mM MgCl2,
60 mM o-mercaptoethanol, 1 mM dATP, dGTP, dTTP and 5 x 10 7M
(Q-32P) dCTP. The mixture is incubated at 42C for 30 min. The product
formed is separated by sephadex G-50 columns and treated with 0.3N
NaOH for 30 minutes at 700C. The 32P cDNAs are annealed with poly A
mRNA of IF mixed with 50 µl of 0.4M sodium phosphate (pH6.8), 0.1%
SDS. The mixture is incubated at 98 C for 5 min followed by annealing.
The DNA-RNA hybrid can be detected by chromatographic separation or
Agarose gel electrophoresis.
• Screening of Recombinant Plasmids: DNA is extracted from the plasmid
containing bacterial cell culture, digested by ECO RI and denatured in alkali.
Followed by denaturation the digested samples are transferred into
nitrocellulose membrane and incubated with 50% formamide, 10 x
Denhardt’s solution, 40 mM Tris-HCl (pH 7.5), 2mM EDTA and 40 µg/ml
yeast RNA for 16 hours at 42°C. The filter papers are washed in SDS and
viewed in transilluminator.
• Construction of Plasmid for Expression of IF: Desired set of primers
are phosphorylated by T4 polynucleotide kinase and radioactive phosphate
in ATP. The primers are further ligated to restriction site complementary
fragment containing the IF cDNA sequence. The mixture is ethanol
precipitated and after subsequent boiling it is cooled to room temperature.
Followed by cooling the mixture is incubated in combined with a 50 µl
solution of 20mM Tris-HCl (pH 7.5), 14 mM MgCl2, 120 mM NaCl,
0.5 mM dATP, dCTP, dGTP, dTTP at 0°C. Followed by this DNA
polymerase I Klenow fragment (10-20 U) are added and incubated at
37°C for 5 hours. The reaction mixture is phenol extracted and restriction
digested with Pst I. The desired product is separated on a 6% agarose gel
and ligated to the desired plasmid for expression.
• Assay for Interferon (IF) Expression in Escherichia coli: The
transformant strain of Escherichia coli containing the expressed IF protein
are grown in LB medium containing 5 µg/ml tetracycline and diluted into
25 ml of M9 medium containing 0.2% glucose, 0.5% casamino acids and
5 mg/ml tetracycline. The cells are pelleted at 500g and the activity of
interferon is detected by cytopathic effect inhibition assay using Sindbis
virus human diploid cells (Refer Figure 7.4).

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NOTES

Fig. 7.4 Schematic Representation of

Recombinant Protein/Drug Production in Bacteria

Applications
The discovery and use of interferon has led to its application as an important
clinical drug to treat various diseases like Japanese encephalitis, autoimmune disease
and hepatitis. More than 13 human genes have been identified which produce
three classes of interferon in cells. The IF-α type is an important activator of
macrophages and dendritic cells against pathogen defence. Various recombinant
forms of IF-α have been produced to treat herpes, chronic hepatitis B, C and
certain cases of malignancy. IFs have also been effective in the treatment of multiple
sclerosis and also exhibit anti-tumour activity. Although IF therapy proves effective
in treating various diseases; it is advised to observe the side effects associated
with every class of IF administered to patients.
7.2.3 Development of Recombinant Vaccines
Usually vaccines contain parts of attenuated or inactivated pathogen proteins serving
as antigens in the body. The development of recombinant vaccines aim at enhancing
the amount of protein produced for commercial and clinical use. The methods and
expression system used has possibilities of high output of various important vaccines.
DNA vaccines are capable of inducing long term immune responses in the cells.
The success of a vaccine lies on the possibility of low antigen variability for the
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Cloning of Human targeted pathogen. However, immunity against the intracellular pathogen has often
Insulin
been difficult to be achieved by the help of conventional vaccines. Reversal of
attenuation is a major risk for the conventional vaccines administered to patients.
Recombinant vaccines negate the risk of toxic adjuncts or reversal issues which
NOTES may occur with the conventional vaccines. Furthermore, sufficient quantity of desired
antigen can be obtained from the recombinant vaccines. The yeast cells are ideal
system for production of recombinant vaccines due to the following features:
• Suitable eukaryotic system capable of functioning for the post-translational
modification of proteins like glycosylation.
• High output of antigen.
• Efficient secretion of antigen into the supernatant and subsequent purification.
Production of Hepatitis B Surface Antigen in Yeast
The production of surface antigen of Hepatitis B in Saccharomyces sp. provides
insights to the application of recombinant vaccine for treatment of Hepatitis B.
This surface antigen protein is a major form present in both glycosylated and non-
glycosylated forms. Although various HB polypeptides have been produced in
Escherichia coli and Bacillus subtilis, the HBs AG form has exhibited higher
antigenecity against the virus. Hybrid expression systems in yeast thus facilitate the
expression of heterologous genes to produce the recombinant form of the protein.
The Yeast alcohol dehydrogenase promoter sequence has been preferably used
for the production of the protein. The promoter usually contains the 5´ flanking
region accompanied by 3´-flanking sequence of the yeast TRP1 gene.
Method of HBs Ag Production in Yeast
Following are the methods of HBs Ag production in yeast (Refer Figure 7.5):
• Enzymes and other Components: Restriction endonucleases, T4 DNA
ligase, and polynucleotide kinases are required for the process of
recombinant plasmid production. DNA polymerase I, S1 endonuclease and
deoxyribonucleotides are essential components for this method
• Plasmid Strains and Growth Conditions: The bacterial transformations
are preferred for Escherichia coli K12 strain and the yeast strain
(20B-12) is preferable for the transformation. Recombinant plasmid with
3-PhosphoGlycerate Kinase (PGK) gene as the 5´ flanking sequence and
contain EcoRI and Pst I restriction sites are produced. The transformed
plasmids present in the Escherichia coli strains are maintained in LB medium
added with 20 µg/ml ampicillin which is used for selection. The transformed
yeast cells are also grown in the selective media of YEPD for obtaining
confirmation of the transformed cells.
• Yeast Transformation and HBs Ag Assay: The plasmids pYeHBs,
pYeHBsd, and YEp13 are commonly used to transform the yeast strains
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 namely XV610-8C, GM3C-2, or 20B-12. The strains are prototrophs for Cloning of Human
Insulin
leucine or tryptophan. The transformed strains are grown in YEPD selection
media. The transformed cells are pelleted down and dissolved in 500 µl
PBS (20 mM sodium phosphate pH 7 and 0.14 M NaCl). The pellet-
buffer homogenate is mixed with glass beads and centrifuged at 5000g in NOTES
low temperature (4°C). The supernant collected can be used for detection
of HBs Ag by ELISA or radioimmuno assay.
• Purification of HBs Ag Particle from Yeast: The yeast cells are
homogenized with glass beads and added to 0.05M Tris HCl at pH 8.0.
The extract is diluted to ten times by adding 5 percent (w/v) in PEG 8000
and 4 percent (w/v) in Dextran 500. The mixture is centrifuged and the top
phase allowed to be separated in a sephadex bead column filtration
chromatography by using NaCl. The antigen obtained can be further purified
by the help of cesium chloride density gradient centrifugation and crystallized.
The form of HBsAg obtained is reconstituted in PBS.
• In-Vivo Labelling and Separation of HBs Ag in Yeast: Transformed
yeast cells growing in the log phase are centrifuged and incubated with 35S
met or 35S cys (radioisotope of cysteine and methionine). The labelled cells
are pelleted to 12,000 g and resuspended in electrophoresis sample buffer
containing (0.1M Tris HCl, pH 6.8, 2 percent SDS, 10 percent glycerol,
5 percent mercaptoethanol, and 0.1 percent bromphenol blue). The presence
of protein is detected by autoradiography after separation in 12%
polyacrylamide gel.

Fig. 7.5 Method of Production of HBsAg Protein in Yeast

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Check Your Progress
1. How assembly of insulin gene done?
NOTES 2. Write a short note on construction of plasmid for expression of A and B
chain of insulin.
3. On what do the success of a vaccine lies?
4. Why are yeast cells ideal system for production of recombinant vaccines?

7.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. The genes for the two polypeptide chains of insulin A and B are engineered
by help of oligonucleotides and T4 DNA ligase which results in the formation
of specific restriction enzyme sites in their structure. This step is necessary
to clone the genes in the pBR322 plasmid. The gene fragments for A and B
chain possess ECORI, BamHI and Hind III restriction sites in their flanking
regions.
2. The pIA1 and pIB1 plasmids are designed to produce the respective
expression vectors for chain A and chain B respectively. The two plasmids
are digested by EcoRI and HindIII enzymes and ligated with the GOI
fragment and most of pBR322 plasmid. Restriction analysis is performed
to check the correct insertion of ampicillin and β-galactosidase gene in the
plasmid.
3. The success of a vaccine lies on the possibility of low antigen variability for
the targeted pathogen.
4. The yeast cells are ideal system for production of recombinant vaccines
due to the following features:
• Suitable eukaryotic system capable of functioning for the post-
translational modification of proteins like glycosylation.
• High output of antigen.
• Efficient secretion of antigen into the supernatant and subsequent
purification.

7.4 SUMMARY

• Gene cloning is an advantageous process which leads to production of

recombinant proteins, hormones and various antigens required for
vaccination.
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• The implication of various plasmids obtained from Escherichia coli facilitates Cloning of Human
Insulin
the process of gene cloning.
• The use of specific set of restriction enzymes and ligases is important to
integrate the Gene Of Interest (GOI) within the plasmid vector designed for NOTES
cloning.
• The efficiency in the production of recombinant proteins appears to be a
beneficial consequence of the completion of human genome project.
• Genentech in the year 1978 originally developed the rDNA mediated
production of human insulin in a commercial scale.
• In the early years of 1970s insulin was extracted from the frozen pancreas
tissues of pigs and cattle.
• A large number of animals were required to cope with the high demand of
this drug which is commonly used as a treatment in diabetics.
• The process of cloning of human insulin gene involved separate cloning of
polypeptide chains into the plasmid followed by the formation of recombinant
protein.
• The plasmid required to clone the GOI is pBR322 which is extracted using
the extraction medium.
• The genes for the two polypeptide chains of insulin A and B are engineered
by help of oligonucleotides and T4 DNA ligase which results in the formation
of specific restriction enzyme sites in their structure.
• The pIA1 and pIB1 plasmids are designed to produce the respective
expression vectors for chain A and chain B, respectively.
• The strains of bacteria which contain the β-galactosidase-insulin A/B chain
produce 30-50% of their cellular protein as the insulin chain.
• The β-galactosidase-insulin hybrid chains are cleaved by cyanogens bromide
and S-sulfonated.
• Interferons were discovered in the year 1957 by A. Issacs and J.
Lindennmann at the National institute of medical research in London.
• The human interferons are a class of homologous proteins necessary for
anti-viral, anti-proliferative and immuno-modulatory function in the cells.
• The name interferon has been derived from their ability to interfere with the
process of viral replication in the infected cells.
• The three classes of interferon (Types I, II and III) are roughly encoded by
around 20 genes identified in animals and humans.
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• Various types of interferon molecules like interferon beta 1a and interferon
beta 1b are implied for the control of diseases like multiple sclerosis and
auto-immune disorders.
NOTES • The first use of artificial human IF as a drug molecule was approved by
Food and Drug Administration in the year 1986.
• The process of IF synthesis in Escherichia coli is accomplished by the
construction of a cDNA library preferably from the mRNA of human
fibroblast IF.
• The mRNA for human IF is transferred into the expression vector of
Escherichia coli to obtain good amount of IF yield.
• The primer fragments are synthesized on the basis of the gene sequence of
the protein (IF).
• The poly-A containing mRNA is extracted from the cell lines using standard
phenol extraction method. The mRNA can be separated and detected by
oligo (dT) cellulose chromatography or gel electrophoresis.
• The cDNA molecules are synthesized with the help of mRNA of IF.
• The cells are pelleted at 500g and the activity of interferon is detected by
cytopathic effect inhibition assay using Sindbis virus human diploid cells.
• The development of recombinant vaccines aim at enhancing the amount of
protein produced for commercial and clinical use.
• Recombinant vaccines negate the risk of toxic adjuncts or reversal issues
which may occur with the conventional vaccines.
• The production of surface antigen of hepatitis B in Saccharomyces sp.
provides insights to the application of recombinant vaccine for treatment of
Hepatitis B.
• The plasmids pYeHBs, pYeHBsd, and YEp13 are commonly used to
transform the yeast strains namely XV610-8C, GM3C-2, or 20B-12. The
strains are prototrophs for leucine or tryptophan.
• The antigen obtained can be further purified by the help of cesium chloride
density gradient centrifugation and crystallized. The form of HBsAg obtained
is reconstituted in PBS.
• The presence of protein is detected by autoradiography after separation in
12% polyacrylamide gel.

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7.5 KEY WORDS Insulin

• Gene cloning: Gene cloning is an advantageous process which leads to

production of recombinant proteins, hormones and various antigens required NOTES
for vaccination.
• Insulin: Insulin is a hormone made by the pancreas that allows your body
to use sugar from carbohydrates in the food that you eat for energy or to
store glucose for future use.

7.6 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is gene cloning?
2. Give the historical perspective of gene cloning.
3. Write a short note on synthesis of interferon in Escherichia coli.
4. Give a diagrammatic representation of recombinant protein/drug production
in bacteria.
Long Answer Questions
1. Define the term recombinant vaccine. Cite an example.
2. Explain the process of cloning and isolation of human insulin.
3. Discuss the method of recombinant interferon production.
4. Elaborate a note on significance of recombinant HBs Ag vaccine production.

7.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
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Cloning of Human Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Insulin
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
NOTES
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 Cloning for Commercial

UNIT 8 CLONING FOR Production of Antibiotics

COMMERCIAL
NOTES
PRODUCTION OF
ANTIBIOTICS
Structure
8.0 Introduction
8.1 Objectives
8.2 Cloning for Commercial Production of Antibiotics
8.3 Answers to Check Your Progress Questions
8.4 Summary
8.5 Key Words
8.6 Self Assessment Questions and Exercises
8.7 Further Readings

8.0 INTRODUCTION

Production of antibiotics is a naturally occurring event that thanks to advances in

science can now be replicated and improved upon in laboratory settings. Due to
the discovery of penicillin by Alexander Fleming, and the efforts of Florey
and Chain in 1938, large-scale, pharmaceutical production of antibiotics has been
made possible. As with the initial discovery of penicillin, most antibiotics have
been discovered as a result of happenstance. Antibiotic production can be grouped
into three methods: natural fermentation, semi-synthetic, and synthetic. As more
and more bacteria continue to develop resistance to currently produced antibiotics,
research and development of new antibiotics continues to be important. In addition
to research and development into the production of new antibiotics, repackaging
delivery systems is important to improving efficacy of the antibiotics that are currently
produced. Improvements to this field have seen the ability to add antibiotics directly
into implanted devices, aerosolization of antibiotics for direct delivery, and
combination of antibiotics with non-antibiotics to improve outcomes. The increase
of antibiotic resistant strains of pathogenic bacteria has led to an increased urgency
for the funding of research and development of antibiotics and a desire for production
of new and better acting antibiotics.
A revolution in industrial microbiology was sparked by the discoveries of
their double-stranded structure of DNA and the development of recombinant DNA
technology. Traditional industrial microbiology was merged with molecular biology
to yield improved recombinant processes for the industrial production of primary
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Production of Antibiotics
and secondary metabolites, protein biopharmaceuticals and industrial enzymes.
Novel genetic techniques such as metabolic engineering, combinatorial biosynthesis
and molecular breeding techniques and their modifications are contributing greatly
NOTES to the development of improved industrial processes. In addition, functional
genomics, proteomics and metabolomics are being exploited for the discovery of
novel valuable small molecules for medicine as well as enzymes for catalysis. The
sequencing of industrial microbial genomes is being carried out which bodes well
for future process improvement and discovery of new industrial products.
In this unit, you will study about cloning for commercial production of
antibiotics in detail.

8.1 OBJECTIVES

After going through this unit, you will be able to:

• Understand the concept of recombinant antibiotic production
• Discuss the efficacy of antibiotic compounds

8.2 CLONING FOR COMMERCIAL PRODUCTION

OF ANTIBIOTICS

The process of genetic manipulation of metabolites has proved advantageous in

clinical and applied research. The concept of cloning of metabolite genes and their
heterologous expression in Escherichia coli result in higher production of
metabolites and natural products. The major challenges faced in the methods of
recombinant DNA technology is the optimisation of suitable protein expression
and obtaining the proteins in their functional conformation. Antibiotic molecules
are small molecular weight compounds which kill or inhibit growth of bacteria
within organisms. Antibiotics are usually produced by various bacteria and fungi
like Penicillium. The major ecological significance of antibiotic production is self-
defence of organism by evading infection. The antibiotics can be extracted from
microbial fermentation and modified enzymatically for clinical and investigational
uses. Recent developments have been successful in cloning the antibiotic
biosynthesis genes in specific trains of Escherichia coli. This shall enable to increase
the commercial production of antibiotics at a larger scale. Advancements in the
field of molecular genetics have opened new perspectives in the improvement of
antibiotic production. The genes encoding enzymes responsible for antibiotic
production have been identified and cloned from the producer organisms. The
plasmids and phage vectors have been implied for the cloning of antibiotic
biosynthesis genes. The DNA of interest can be successfully transferred to the
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protoplast of Streptomyces and Escherichia coli. Heterologous expression of Cloning for Commercial
Production of Antibiotics
genes in Escherichia coli has been successful by using the effective promoters of
the bacteria. Promoter-probe vectors have been constructed to produce the gene
clones for transcriptional elements containing control signals. Genetic manipulation
NOTES
has lead to the production of high activity biosynthesising enzymes for antibiotic
pathway. Creation of new genetic recombinations likely produces antibiotic
molecules with new biochemical properties.
In the last few decades significant development has occurred in the field of
genetic recombination studies which have provided flourishment of commercial
antibiotics. The methods of genetic engineering have led to the production of
antibiotic compounds within recombinant cells converted to producer types.
Protoplast fusion method has been applied for mutants of a streptomycin producer
(Streptomyces griseus) and an istamycin producer (Streptomyces tenjimariensis)
which resulted in the production of a new hybrid strain producing the novel antibiotic
compounds like indolizomycin. Similar methods have resulted in the formation of
rifamycin derivates (16, 17-dihydrorifamycin S and 16, 17-dihydro-17-hydroxy-
rifamycin S) in hybrid strains of Nocardia mediterranei. Recombination among
various species of Sterptomyces has led to the production of antibiotics which are
different from that of parental strains. Combinatorial biosynthesis approach
associated with the heterologous expression of antibiotic molecules in recombinant
vectors has gained significant advancements. The main approaches implied in this
context are associated with the detailed investigation of biosynthesis genes and
metabolic pathways. The genetic recombination method includes:
• Targeted gene disruption.
• Insertion of multiple copies of a positive regulator gene by cloning.
This process liberates novel biomolecules effective as antibiotics. New forms
of erythromycin have been produced by inducing mutation in the synthase encoding
genes from Saccharopolyspora erythrea. Interestingly, a three plasmid
recombinant system has been developed for the heterologous expression of 6-
deoxyerythronolide B synthase gene. This method led to formation of different
polyketide lactones in Streptomyces lividans. The recombinants were produced
by replacing erythromycin producing genes by rafamycin. Methymycin and
pikromycin have been produced in a modified form by cloning of a gene encoding
calicheamicin obtained from Micromonospora echinospora subsp. Calichensis.
The cloning of the gene resulted in production of Tetracenomycin C in new species
of Streptomyces. Tetracenomycins have been produced by the introduction of 25
kb cosmid from the elloramycin biosynthesis pathway of S. olivaceus which was
introduced into a mutant of urdamycin producer S. fradiae. The cosmid contains
a gene encoding the enzyme glycosyltransferase gene.
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Production of Antibiotics
Various antibiotic substances have been produced in the last decades by the help
of recombinant DNA technology. The methods implied mainly aim to increase
NOTES production of the compounds by enhancing the activity of the biosynthesizing
enzymes. Furthermore, the structural modifications of many antibiotic molecules
have been reported to modulate its efficiency.
Echinomycin: The cloning of non-ribosomal peptide echinomycin from
Steptomyces has been successfully performed in specific strains of Escherichia
coli. The monocistronic ECM Gene (Extracellular Matrix Gene) transferred to a
expression plasmid is responsible for the product called echinomycin. This process
has also been associated with the genetic manipulations of the nature of the antibiotic.
This process has also been associated with the formation of triostin and S-
adenosylmethionine associated product. There lie certain differences between
genetic engineering and metabolic engineering of antibiotic production (Refer Figure
8.1). In the later case the metabolic and cellular consideration are taken into account
for proper expression of the metabolite.
Table 8.1 Instances of Antibiotic Production by
Heterologous Expression and Metabolic Engineering

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Production of Antibiotics

NOTES

Fig. 8.1 Metabolic Engineering of Echinomycin A Biosynthesis

Gene and Production of Triostin A

β-Lactams: These antibiotics are associated with the chemotherapy of bacterial

infections and mainly include Penicillin G, Cephalosporin C, Cephamycin C.
Cephalosporin C: The production of Cephalosporin C has been achieved by
construction of recombinant strain produced by protoplast fusion of Acremonium
chrysogenum. The low titer producing strain was supplemented by high levels of
methionine after its protoplast fusion with a high titer producing strain. The new
recombinant strain produced was of sporulating type and produced about 40%
more antibiotic than the non-recombinant types. Multiple copies of cyclase gene
were inserted into Acremonium chrysogenum which improved Cephalosporin C
production. An industrial strain of Acremonium chrysogenum 394-4 was
transformed with plasmid having pcbC and the cefEF gene obtained from a mutant
line. The transformant contained 50% more Cephalosporic C in comparison with
the non-transformants. Acetyl transferase, expandase and hydroxylase genes have
been cloned and inserted in multiple copies within the transformants. This resulted
in increased antibiotic production.
Cephamycin C: The production of Cephamycin has also been increased by the
methods of protoplast fusion. The recombinant strain produced from single
mutagenesis treatment was observed to possess capacity of increased yield. The
improved Cephamycin C producing strain have been derived from Nocardia
lactamdurans which resulted in 10-15% more yield. The genetic manipulation of
cca-R gene from Streptomyces clavuligerus which functions as a positive regulator
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Cloning for Commercial of cephamycin pathway resulted in overproduction of its protein. The lat gene
Production of Antibiotics
which encodes for lysine-aminotransferase also resulted in the overproduction of
antibiotic.
Clavulanic Acid: The biosynthesis of this antibiotic initiates from the condensation
NOTES
reaction between by L-arginine and D-Glyceraldehyde-3-Phosphate (G3P).
Development of certain strains has improved the production of clavulanic acid.
The enhancement was found to be around tenfold to that of the wild types. The
recombinant strain of S. clavuligerus NRRL 38 has been reported to exhibit
higher production of this antibiotic. The G3P amount appears to be limiting in
nature. Thus, targeted disruption of G3P dehydrogenase encoding genes- Gap 1
and Gap 2 has resulted in the increase of antibiotic production. Similar response
was obtained as a result of overexpression of the positive regulatory genes.
Semisynthetic Cephalosporin: This molecule is chemically 7-
AminoCephalosporanic Acid (7-ACA) or 7-AminoDeaCetoxycepAlosporanic
Acid (7-ADCA). Earlier methods of production of this molecule involved the use
of toxic reagents, which, however, has now been replaced by coning and genetic
engineering methods. Genetic recombinants have been created for P. chrysogenum
which have been transformed with Streptomyces lipmanii cefD and S. clavuligerus
cefE genes which encode for the intermediate form of the antibiotic
DeAcetOxyCephalosporin C (DAOC) at moderate titers. The cloning method
involving cefE gene from S. clavuligerus or cefEF and cefG from A. chrysogenum
transformed into P. chrysogenum has led to the production of effective intermediate
compounds having properties of cephalosporin. Moderate titers of Penicillin N
have been produced by one step gene replacement for cefEF from the industrial
Cephalosporin C production strain of A. chrysogenum. Escherichia coli strains
have been created by transformation of D-amino acid oxidase gene from Trigonopsis
variabilis and the glutaryl-7-aminocephalosporanic acid acylase gene from
Pseudomonas sp. This strain of Escherichia coli has been effective in producing
derivatives of cephalosporin.
Non-β-Lactam Antibiotics: Genetic engineering method has been implied to
produce non-β-lactam antibiotics like Daptomycin. Transposition mutants of
Streptomyces roseosporus Tn 5099 have been reported to produce 55-60%
higher daptomycin in comparison with the non-transformants.
Cloning Method Approach to Optimize Penicillin Production
Penicillin is an important antibiotic having potential clinical applications against
several diseases. The antibiotic leads to acylation reaction in the active site of
D-alanine carboxypeptidase an important enzyme necessary to complete the
formation of cell wall in bacteria. This important enzyme catalyzes the crosslinking
of peptide-glycan layer of cell wall. This antibiotic is a structural analog of the
acyl-D-alanyl-D-alanine end of the penta-peptide side chain of the growing
peptidoglycan chain. Chromatographic separation coupled to binding assays have
thus revealed that penicillin and the substrate of D-alanine carboxypeptidase
compete with each other and it former cause acylation in the active site of the
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enzyme. Thus penicillin G acylase is one of the important enzymes necessary for Cloning for Commercial
Production of Antibiotics
the optimization of penicillin action in cells.
Protoplast Fusion has resulted in the improvement in the production of Penicillin
from the overproducing strains of Penicillium chrysogenum. Repeated back cross
NOTES
of the low yield strain with that of high producing strain resulted in 60% increase in
production of the antibiotic. Cloning and insertion of multiple copies of the
biosynthesis genes resulted in higher levels of transcription within the recombinant
cells.
Gene Overexpression: Penicillin overproduction was possible by overexpression
of phenylacetic acid-activating CoA ligase derived from Pseudomonas putida.
The ethanol dehydrogenase promoter was applied to overexpress this gene in
Aspergillus nidulans. This heterologous expression resulted in 30-fold increase
in the antibiotic production.
Cloning and Overexpression of Penicillin G Acylase in Escherichia coli:
Penicillin G Acylase (PGA) is an important enzyme responsible for the biosynthesis
of semi-synthetic penicillin. The natural strains of Escherichia coli are selected to
create recombinant expression vectors necessary to overproduce Penicillin. The
primer is designed based on the gene sequence of PGA and cloned to insert in the
recombinant strains of Escherichia coli using T7 promoter for expression. The
cloning method is successful in pGEM -T easy vector which results in the
overexpression of PGA in Escherichia coli.
Cloning and Overexpression of Penicillin G Amidase: Penicillin G
Amidase (EC 3.5.1.11; PAC) catalyzes the conversion of Penicillin G to
phenylacetic acid and 6-aminopenicillanic acid which triggers the synthesis of
β-lactam antibiotics. Various strains of Gran-Positive and Gam-Negative Bacteria,
such as Alcaligenes faecalis (ATCC 19018), Arthrobacter viscosus (ATCC
15294), Bacillus megaterium (ATCC 14954), Escherichia coli (ATCC 11105)
and Kluyvera citrophila (ATCC21285). Interestingly, to improve the efficiency
of the enzyme Pichia pastoris (methylotrophic yeast) has been used to overexpress
the enzyme. This eukaryotic system is favourable in terms of post transational
modification of proteins. P. pastoris has also been used as an ideal expression
system for obtaining proper functional enzyme. Escherichia coli has been effectively
used to produce the enzyme from Alcaligenes faecalis. Bioreactors have been
designed with fermentation reaction and substrate of rhamnose which have produced
up to 4500 U of the enzyme.
Cloning and Heterologous Expression of Penicillin Biosynthetic Genes:
Interesting advancements have been achieved for the heterologous expression of
genes associated with penicillin biosynthesis from Penicillium chrysogenum. The
cosmid clones obtained for the Penicillin biosynthetic genes are transformed to
Neurospora crassa and Aspergillus niger. The major enzymes encoded by the
Penicillin biosynthesis genes are namely δ-(L-α-aminoadipyl)-L-Cysteinyl D-Valine
Synthetase (ACVS), IsoPenicillin N Synthetase (IPNS) and acyl coenzyme A: 6-
AminopeniCillanic acid acyltransferase (ACT).
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Cloning for Commercial Cloning and Overexpression of Isopenicillin-N-Acyltransferase: Certain
Production of Antibiotics
recombinant strains of Penicillium chrysogenum have been reported to yield
high titers of Penicillin. Cloning method has been implied to induce multiple copies
of the Penicillin biosynthesis genes namely δ-(l-α-Aminoadipyl)-L-Cysteinyl-D-
NOTES Valine Synthetase (ACVS), IsoPenicillin N Synthase (IPNS), and Isopenicillin N
AcylTransferase (IAT). IAT expression in sufficient amount is a major limiting factor
for penicillin biosynthesis. Higher expression and elevated activity of IAT produces
optimum amount of penicillin. Methylotrophic yeast Hansenula polymorpha has
been implied for successful execution of the Penicillin biosynthetic pathway. Yeast
systems are suitable expression systems rendering the proper functional form of
the enzyme.
Conclusions
Application of microbial genomics has resulted in the identification of various novel
antibiotics. Novel discoveries in the field of chemical science associated with genome
mining, study of gene sequences and heterologous expression are some of the
approaches towards producing commercial antibiotics. The process of genome
mining facilitates to use DNA sequences and bioinformatics in indentifying the
gene clusters of antibiotic producing organisms. The current state of knowledge
on antibiotics has revealed that around 25 genes out of 2700 pathogenic traits are
targets for antibiotics. Comparison between the pathogenic and non-pathogenic
forms of bacteria has revealed the percentages of gene pools which are potential
targets of various antibiotics.

Check Your Progress

1. What does the concept of cloning of metabolite genes and their heterologous
expression depends upon?
2. What are the major challenges faced in the methods of recombinant DNA
technology?
3. What is the major ecological significance of antibiotic production?
4. List the methods included in genetic recombination.
5. What is non-β-Lactam antibiotics?
6. How is protoplast fusion useful?
7. Write short note on gene overexpression.

8.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. The concept of cloning of metabolite genes and their heterologous expression

in Escherichia coli result in higher production of metabolites and natural
products.
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 2. The major challenges faced in the methods of recombinant DNA technology Cloning for Commercial
Production of Antibiotics
is the optimisation of suitable protein expression and obtaining the proteins
in their functional conformation.
3. The major ecological significance of antibiotic production is self-defence of
NOTES
organism by evading infection.
4. The genetic recombination method includes:
• Targeted gene disruption.
• Insertion of multiple copies of a positive regulator gene by cloning.
5. Non-β-Lactam antibiotics uses the Genetic engineering method to produce
non-β-lactam antibiotics like Daptomycin. Transposition mutants of
Streptomyces roseosporus Tn 5099 have been reported to produce 55-
60% higher Daptomycin in comparison with the non-transformants.
6. Protoplast fusion has resulted in the improvement in the production of
Penicillin from the overproducing strains of Penicillium chrysogenum.
Repeated back cross of the low yield strain with that of high producing
strain resulted in 60% increase in production of the antibiotic. Cloning and
insertion of multiple copies of the biosynthesis genes resulted in higher levels
of transcription within the recombinant cells.
7. Gene overexpression refers to the Penicillin overproduction which was
possible by overexpression of phenylacetic acid-activating CoA ligase derived
from Pseudomonas putida. The ethanol dehydrogenase promoter was
applied to overexpress this gene in Aspergillus nidulans. This heterologous
expression resulted in 30-fold increase in the antibiotic production.

8.4 SUMMARY

• The concept of cloning of metabolite genes and their heterologous expression

in Escherichia coli.
• The major challenges faced in the methods of recombinant DNA technology
are the optimisation of suitable protein expression and obtaining the proteins
in their functional conformation.
• Antibiotics are usually produced by various bacteria (Bacillus,
Streptomyces), actinomycetes (Streptomyces, Actinomadura,
Actinoplanes, Micromonospora) and fungi like Penicillium.
• The antibiotics can be extracted from microbial fermentation and modified
enzymatically for clinical and investigational uses.
• The genes encoding enzymes responsible for antibiotic production have
been identified and cloned from the producer organisms.
• The plasmids and phage vectors have been implied for the cloning of
antibiotic biosynthesis genes.
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Cloning for Commercial • Heterologous expression of genes in Escherichia coli has been successful
Production of Antibiotics
by using the effective promoters of the bacteria.
• Genetic manipulation has lead to the production of high activity
biosynthesising enzymes for antibiotic pathway.
NOTES
• Creation of new genetic recombinations likely produces antibiotic molecules
with new biochemical properties.
• Recombination among various species of Sterptomyces has led to the
production of antibiotics which are different from that of parental strains.
• Combinatorial biosynthesis approach associated with the heterologous
expression of antibiotic molecules in recombinant vectors has gained
significant advancements.
• The genetic recombination method includes, the targeted gene disruption
and the insertion of multiple copies of a positive regulator gene by cloning.
• Methymycin and pikromycin have been produced in a modified form by
cloning of a gene encoding calicheamicin obtained from Micromonospora
echinospora subsp. Calichensis.
• The cloning of non-ribosomal peptide echinomycin from Steptomyces has
been successfully performed in specific strains of Escherichia coli.
• The production of cephalosporin C has been achieved by construction of
recombinant strain produced by protoplast fusion of Acremonium
chrysogenum.
• The new recombinant strain produced was of sporulating type and produced
about 40% more antibiotic than the non-recombinant types.
• The production of cephamycin has also been increased by the methods of
protoplast fusion.
• The genetic manipulation of cca-R gene from Streptomyces clavuligerus
which functions as a positive regulator of cephamycin pathway resulted in
overproduction of its protein.
• Genetic engineering method has been implied to produce non-β-lactam
antibiotics like Daptomycin.
• Transposition mutants of Streptomyces roseosporus Tn 5099 have been
reported to produce 55-60% higher Daptomycin in comparison with the
non-transformants.
• Penicillin is an important antibiotic having potential clinical applications against
several diseases.
• The antibiotic leads to acylation reaction in the active site of D-alanine
carboxypeptidase an important enzyme necessary to complete the formation
of cell wall in bacteria.
• Penicillin G Acylase is one of the important enzymes necessary for the
optimization of Penicillin action in cells.
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 • Protoplast fusion has resulted in the improvement in the production of Cloning for Commercial
Production of Antibiotics
penicillin from the overproducing strains of Penicillium chrysogenum,
• Penicillin overproduction was possible by overexpression of phenylacetic
acid-activating CoA ligase derived from Pseudomonas putida.
NOTES
• The natural strains of Escherichia coli are selected to create recombinant
expression vectors necessary to overproduce Penicillin.
• Penicillin G Amidase (EC 3.5.1.11; PAC) catalyzes the conversion of
Penicillin G to phenylacetic acid and 6-aminopenicillanic acid which triggers
the synthesis of β-lactam antibiotics.
• Interesting advancements have been achieved for the heterologous
expression of genes associated with penicillin biosynthesis from Penicillium
chrysogenum.
• Yeast systems are suitable expression systems rendering the proper functional
form of the enzyme.
• The process of genome mining facilitates to use DNA sequences and
bioinformatics in indentifying the gene clusters of antibiotic producing
organisms.
• The current state of knowledge on antibiotics has revealed that around 25
genes out of 2700 pathogenic traits are targets for antibiotics.

8.5 KEY WORDS

• Antibiotic molecules: Antibiotic molecules are small molecular weight

compounds which kill or inhibit growth of bacteria within organisms.
• β-lactams: Antibiotics associated with the chemotherapy of bacterial
infections that mainly includes Penicillin G, Cephalosporin C, Cephamycin
C.
• Penicillin: It is an important antibiotic having potential clinical applications
against several diseases.

8.6 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. Write a short note on cloning methods for production of various antibiotics.
2. What is clavulanic acid?
3. Explain semisynthetic cephalosporin.
4. What is non-β-lactam antibiotics?
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Cloning for Commercial 5. Give the cloning method approach to optimize Penicillin production.
Production of Antibiotics
Long Answer Questions
1. Define metabolic engineering. Explain its significance in the production of
NOTES recombinant antibiotics.
2. Explain with the help of table instances of antibiotic production by
heterologous expression and metabolic engineering.
3. Explain the various strategies adopted through cloning method for Penicillin
production.
4. What do you mean by heterologous expression? Explain its role in
production of recombinant antibiotics.

8.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 Cloning Methodologies

UNIT 9 CLONING
METHODOLOGIES
NOTES
Structure
9.0 Introduction
9.1 Objectives
9.2 Cloning Methodologies
9.2.1 α (Alpha) Complementation Test
9.2.2 Sticky and Blunt End Cloning
9.2.3 Blunt End Cloning Method
9.2.4 Cloning From mRNA
9.2.5 Cloning from Genomic DNA Library
9.2.6 Cloning of Genomic DNA
9.2.7 Shotgun Cloning
9.3 Answers to Check Your Progress Questions
9.4 Summary
9.5 Key Words
9.6 Self Assessment Questions and Exercises
9.7 Further Readings

9.0 INTRODUCTION

Cloning is the process of producing genetically identical individuals of

an organism either naturally or artificially. In nature, many organisms produce clones
through asexual reproduction. Cloning in biotechnology refers to the process of
creating clones of organisms or copies of cells or DNA fragments (molecular
cloning). Beyond biology, the term refers to the production of multiple copies
of digital media or software.
The term clone, coined by Herbert J. Webber, is derived from the Ancient
Greek word ‘twig’, referring to the process whereby a new plant can be created
from a twig. In botany, the term ‘lusus’ was traditionally used. In horticulture, the
spelling ‘clon’ was used until the twentieth century; the final e came into use to
indicate the vowel is a ‘long o’ instead of a ‘short o’. Since the term entered the
popular lexicon in a more general context, the spelling clone has been used
exclusively.
Molecular cloning is a set of experimental methods that are used to assemble
recombinant DNA molecules and to direct their replication within host organisms.
The use of the word cloning refers to the fact that the method involves the replication
of one molecule to produce a population of cells with identical DNA molecules.
Molecular cloning generally uses DNA sequences from two different organisms -
the species that is the source of the DNA to be cloned, and the species that will
serve as the living host for replication of the recombinant DNA. Here the whole
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Cloning Methodologies process is outlined using the example of cloning by restriction and ligation, which
is the traditional method.
In this unit, you will study about cloning methodologies which includes a
complementation, sticky and blunt end cloning, cloning from mRNA, synthesis of
NOTES
cDNA, cloning cDNA, cDNA library, cloning from genomic DNA, genomic library,
and shotgun cloning in detail.

9.1 OBJECTIVES

After going through this unit, you will be able to:

• Discuss various methods of cloning
• Analyse α complementation
• Elaborate on the synthesis of cDNA
• Understand the concept of cDNA library
• Discuss the cloning from genomic DNA and genomic library
• Explain the method of shotgun cloning

9.2 CLONING METHODOLOGIES

Cloning is the process of taking genetic information from one living thing and creating
identical copies of it. The copied material is called a clone. Geneticists have cloned
cells, tissues, genes and entire animals. Although this process may seem futuristic,
nature has been doing it for millions of years. For example, identical twins have
almost identical DNA, and asexual reproduction in some plants and organisms
can produce genetically identical offspring. And scientists make genetic doubles in
the lab, though the process is a little different.
Following are some of the methods of cloning that are discussed below:
9.2.1 α (Alpha) Complementation Test
The method of α (alpha) complementation helps in screening of recombinant
plasmids present in the bacterial cells which contain the gene of interest inserted
into the plasmid. This method can distinguish the bacterial colonies which possess
only plasmids while the ones which contain plasmids with the GOI integrated in
them.
The method is based on the assay of an enzyme called β-galactosidase
acting in the substrate X Gal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside).
Due to reasons of the differences appearing in the colour of colonies, the method
is known as blue-white screening method. Thus, success of ligation in vector-
based cloning methods can be confirmed by this complementation test. The DNA
of interest is integrated into the vector and transformed into the Escherichia coli
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cells for cloning. The transformed cells of the bacteria are preferentially grown in Cloning Methodologies

the presence of X Gal in the growth medium. The recombinant cells contain vectors
integrated with the gene of interest will appear white in nature while the non-
transformed colonies will appear blue in colour. X Gal is a widely used substrate
used for assaying the activity of the enzyme β-galactosidase. X Gal is chemically NOTES
an indoxyl glycoside ester molecule which upon hydrolysis by the enzyme produces
a blue compound. The β-galactosidase enzyme is structurally a homotetramer in
its active state which is encoded by the lacZ gene of the lac operon.
Functioning of α Complementation Method
The β-galactosidase enzyme in its mutant form contains the N-terminal residues
11—41 deleted and is termed as the omega-peptide. This mutant form of the
enzyme is inactive and unable to form the functional homotetramer. However, in
the presence of the peptide (N-terminal fragment of the protein) the mutated form
of the enzyme can be reverted to the functional homotetramer form. The test is
performed by using M13 mutant Escherichia coli. In-vitro addition of cyanogen
bromide can restore the activity of the enzyme. The method involves the non-
functional parts of the protein, i.e., the host Escherichia coli contains the lacZ
deletion mutant (lacZ M15) and contains the omega peptide. The vectors contain
the lacZα sequence encodes the first 59 residues of the enzyme. In the process of
complementation the vector with lacZα sequence is being transformed into the
lacZ M15 cells. This process of complementation results in the restoration of
the functional enzyme. The wild type form of the enzyme is encoded by a gene of
3.1 kb fragment. The screening method for detecting the transformed colonies
functions by disruption of the α-complementation process. A Multiple Cloning
Sites (MCS) is present in the lacZα sequence of the vector. The MCS site contains
certain restriction enzyme target sites. The foreign gene can be inserted into the
MCS site thus leading to the formation of α-peptide. Thus, the vectors containing
the GOI inserted are incapable of forming the functional β-galactosidase enzyme.
Detection of Transformants by Complementation Method
The assay of the active form of β-galactosidase enzyme is possible by using X Gal
in an agar plate. The catalytic activity of β-galactosidase enzyme cleaves X Gal to
produce 5-bromo-4-chloro-indoxy which upon dimerization and oxidation leads
to formation of a blue coloured insoluble compound called 5, 5'-dibromo-4, 4'-
dichloro-indigo. Thus, the Escherichia coli cells which contain functional
β-galactosidase enzyme appear blue in colour. Thus, blue colour indicate that the
vectors in the cells of these colonies contain uninterrupted lacZα (without gene
insert), while the white colonies indicate that X Gal has not been hydrolyzed due
to the absence of functional β-galactosidase enzyme (Refer Figure 9.1). The white
colonies contain interrupted lacZα gene in the vector due to the presence of gene
inserted in it. Thus white colonies represent transformants with GOI incorporated
in the vector. This method requires the combination of correct vector and competent
cells. Vectors like pUC19 and pBluescript are usually used for the presence of
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Cloning Methodologies lacZα gene. The Escherichia coli cells should contain the mutant form of lacZ
gene so that its function is complemented by the vector mediated lacZ gene product.
Some of the commonly used Escherichia coli strains are JM109, DH5α, and
XL1-Blue.
NOTES
Limitations of the Method
There are certain conditions when the blue and white colonies may exhibit false
results due to the following reasons:
• White colonies may not contain the desired recombinant vector. This may
be due to reasons like the ligated DNA may not be the correct one. Some
linearized vectors may be ligated in such a form that lacZα product is not
produced and the colonies appear white.
• Certain white colonies may not contain any vector and appear as satellite
colonies after the depletion of the antibiotic in the medium.
• It may be possible that certain blue colonies may contain the insert in frame
with the lacZα gene and the insert lacks a STOP codon. This will result in
formation of a fusion protein with functional activity similar to lacZα enzyme.
• Recombinant constructs may sometime lead to formation of light blue
colonies which may complicate its identification.

Fig. 9.1 Reaction Catalyzed by the Activity of β-Galactosidase

Enzyme on the Substrate X Gal

Fig. 9.2 Blue White Colonies Appearing as a Result of Complementation Test and
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178 Material
9.2.2 Sticky and Blunt End Cloning Cloning Methodologies

Cloning of double stranded DNA into the vector appears to be the most important
step in the methodological steps of molecular biology. The process of gene cloning
is associated with sequencing, preparing DNA libraries and in expression of coding NOTES
and non-coding RNA.
Sticky or Cohesive End Cloning
This method of cloning involves the formation of overhanging fragments at the two
termini of dsDNA and vector which are then ligated together. The ligation is based
on the prevalence of hydrogen bond formation between the two sticky ends of
GOI and vector molecule. The restriction enzymes cleaved the dsDNA at the
respective recognition sequences. In many cases the restriction enzymes produce
staggered ends with 3´ or 5´ overhanging sequences which can be, however, joined
by ligases to reform the entire sequence. To accomplish the process of sticky end
cloning both the dsDNA and the vector are digested with the restriction enzyme,
so as to produce the 3´/5´ overhangs of 1-4 nucleotide long. These overhangs are
thus referred to as the sticky ends. The enzymes used for the 3´ and 5´ end should
have their sites of recognition preferably more than 10 nucleotide apart. The
specificity of the restriction enzymes regulates the directionality of the integration
of the gene insert. Furthermore, the involvement of hydrogen bonding makes the
process more efficient in comparison with blunt end cloning.
Features of Sticky End Cloning
• The restriction enzyme cleaving sequence should be preferably present both
in the insert sequence and in the Multiple Cloning Sites (MCS) of the vector.
However, this sequence should not exist in other regions of the two DNA
molecules.
• The insert and the vector are digested in two separate reactions by using the
same restriction enzyme.
• Followed by digestion, the plasmid is dephosphorylated and both the insert
and vector are purified from the enzyme mixture.
• The insert is then ligated into the vector and transformed into Escherichia
coli for cloning.
• The cloning plasmid should desirably possess the features like Origin of
Replication (Ori site), Multiple Cloning Site (MCS) possessing the restriction
enzyme sequences. Furthermore, the plasmid should contain the antibiotic
resistance gene necessary for selection of transformed plasmid containing
Escherichia coli cells.
• The method of sticky end cloning involves ligation of the insert into the vector
which recreates the restriction sites. This feature enables the screening of
clones or for adding an additional fragment of DNA in the successive steps
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Cloning Methodologies • The cleaving of dsDNA by the restriction enzyme may occur at or near to
the restriction recognition sequence. Thus, to enable better binding of DNA
to the enzyme prior to binding additional nucleotide sequences called buffer
are added at the end of the insert. The length of the buffer fragment added
NOTES depends upon the nature of the restriction enzyme used.
• The multiple restriction sites present in the MCS of the vector should not be
placed at direct vicinities but preferably separated by 12 bp buffer.
• Various methods like heat inactivation or purification by column, gel or phenol
extraction is necessary for terminating the reaction of restriction enzyme.
• The concentration of ions present in the reaction mixture along with the
quantity of buffer appears to be an important determinant of enzyme activity.
• The blunt ends of the vectors (if any) produced by restriction digestion are
required to be dephosphorylated. This process is accomplished by the
application of alkaline phosphatase enzyme. This results in the prevention
of relegation of vector without addition of the insert.
• Gel purification of the vector is important to purify it form any uncut vectors,
short DNA fragments and restriction enzymes.
Methodology for Sticky End Cloning
• Digestion of vector DNA is performed by the use of two different restriction
enzymes. Followed by digestion the ends of the vector are dephosphorylated
using calf-intestine or shrimp alkaline phosphatase enzyme. This step reduces
the chance of non-recombinants formed due to self-ligating vectors.
• The digested vector is purified by agarose gel electrophoresis which omits
short DNA fragments, residual nicked or supercoiled vector DNA.
• The gene insert is digested by restriction enzyme and purified by agarose
gel electrophoresis.
• The process of ligation of insert and vector involves the formation of phospho-
diester bond between the adjacent 5´-phosphate and 3´-hydroxyl residues.
The enzymes implied for the catalysis of ligation are commonly Escherichia
coli DNA ligase or T4 DNA ligase. The later is preferred for its ability to
join blunt end DNA fragments.
• The ligation step is an ATP dependent process carried out at a temperature
of 4-25°C. The annealing temperature of cohesive end having short termini
is at a range lower than room temperature.
• The concentration of T4 DNA ligase, vector and insert are important to
obtain the success in the ligation step. Alteration in the ratio of the molecules
may result in formation of secondary structures like concatemers. A
concatemer is a long continuous DNA molecule that contains multiple copies
of the same DNA sequence linked in series.
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• The cloning method should be preferably executed by using commercially Cloning Methodologies

available Escherichia coli strain where the methyltransferases Dam (DNA

adenine methyltransferase) and Dcm (DNA cytosine methyltransferase) are
deactivated. These enzymes, if active may methylate the restriction sites,
thus preventing the activity of restriction enzymes. NOTES
• Plasmid containing bacteria are preferably stored in 15% glycerol at a
temperature of -70°C. Addition of EDTA leads to chelating of Mg2+ which
prevents nuclease activity (Refer Figures 9.3 and 9.4).

Fig. 9.3 Method of Sticky End Cloning

Fig. 9.4 Adaptor Molecules Attached to Prevent Self Ligation of Vectors

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Cloning Methodologies 9.2.3 Blunt End Cloning Method
The blunt end method is executed for integration of dsDNA into the linearized
plasmid vector where both the fragments possess blunt ends without any
NOTES overhanging termini at their ends. This method does not involve hydrogen bond
stabilization from overhanging complementary bases, but involves the transient
union of 5´ phosphate and 3´ hydroxyl groups joined together by the help of T4
DNA ligase enzyme. This method appears to be one of the convenient process of
cloning of dsDNA into the vector. Unlike cohesive or sticky end cloning method it
does not involve any enzymatic digestion and purification steps associated with
blunt end method. The chances of success of integration of dsDNA into the vector
facilitated by blunt end method possess fewer sequence limitations in comparison
with other methods (Refer Figure 9.5).

Fig. 9.5 Method of Blunt End Cloning

Features of Blunt End Cloning Method

• This method appears around 10-100 times less efficient than sticky end
method. This inefficiency results due to absence of colonies or wrong insertion
of gene of interest in the recombinant vectors.
• Tendencies of vectors being re-circularized lead to failure in the integration
of GOI (Gene Of Interest). This results in the presence of empty vectors.
Thus prior to sequence verification the vectors should be screened by
restriction digestion or PCR amplification.
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 • A major advantage of the blunt method is that it does not require any Cloning Methodologies

restriction digestion site in its sequence. Thus, this method excludes any
unwanted sequence additions which might complicate the process. This
method therefore appears to be executed comparatively at a faster rate.
NOTES
Methodology for Blunt End Cloning
• Vectors are preferably prepared by digesting the DNA at the Multiple
Cloning Sites (MCS) which contains restriction digestion sites. This step
results in the formation of blunt ends.
• Restriction sites which produce overhanging ends can also be implied which
is subsequently removed or filled by blunt ends. This method can, however,
be avoided because the success of blunting reaction is often difficult to
access.
• Linear plasmids can be produced by PCR by using 5´ primers containing
complementary sequence of the insertion region. Detection of linear plasmid
can be performed by agarose gel electrophoresis where it appears as a
distinct band. The supercoiled plasmids usually represent smears. The
plasmid can be dephosphorylated for ligation and amplification.
• The blunt end of the gene of interest may be required to be phosphorylated.
Annealing of oligonucleotides, if required, is desired to contain 5´ phosphate.
• Commercially available T4 polynucleotide kinase enzyme may be used to
attach phosphates to 5´ ends of dsDNA.
• During certain cases the ends of the insert may require to be converted to
blunt ends. Sometimes Taq polymerase reactions lead to the presence of an
overhanging adenine at the 3´ end. The similar situation may appear as a
result of shearing or sonication.
• Inserts produced by restriction digestion or by annealing of oligonucleotides
may also be required to be converted to blunt ends.
• T4 DNA polymerase or Klenow fragment of DNA polymerase I can be
used to remove DNA overhangs from the gene inserts. Klenow is a protein
fragment derived from Escherichia coli DNA polymerase.
• Ligations for blunt end cloning method require the involvement of 5´ phosphate
groups and 3´ hydroxyl groups which is more transient than that in sticky
end cloning.
• Blunt end method does not involve hydrogen bond stabilization and are
consequently more sensitive to the reaction components and their
concentrations.
• The chances of success of insertion of GOI by using blunt end method
depends upon the concentration of insert.
• Under certain condition s, intramolecular circularization of plasmid may be
associated with integration of multiple inserts thus forming concatemers. Self-Instructional
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Cloning Methodologies • Concentration of T4 DNA ligase influences the efficiency of blunt end ligation.
• An alternative method of blunt end ligation involves a reaction mixture of
plasmid and inserts containing blunt end producing restriction enzyme and
DNA ligase.
NOTES
9.2.4 Cloning From mRNA
cDNA molecules represent the mRNA population of a tissue and represents the
diversity of the spliced isoforms of mRNA. The cellular mRNA is reverse
transcribed to form the cDNA fragment. The cloned cDNA fragments in eukaryotes
lack the presence of introns and non-coding parts of the DNA. Usually bacterial
genes unlike eukaryotes do not contain intron regions. cDNA fragments are smaller
in size than the gene fragment present in the genomic DNA. cDNA preparation
from mRNA is followed by screening of particular clones. Thus a cDNA library is
the representative of the RNA population of the tissue. Thus, unlike genomic DNA
the pool of cDNA for a tissue is variable at different developmental stages of the
tissue. The phenomenon of alternate splicing results in the formation of cDNA
formed from differentially spliced fragments. The abundance of mRNA differs
according to the nature and developmental stage of the tissue. The isolation of
cDNA clones requires the development of cDNA libraries. Lambda vectors are
suitable for obtaining cDNA clones. Enhancement of mRNA production prior to
construction of cDNA libraries.
Synthesis of cDNA
The process of cDNA Synthesis involves three major steps namely:
• First Strand DNA Synthesis from the mRNA Strand by the activity of
Reverse Transcriptase.
• Removal of RNA Strand.
• Second Strand DNA Synthesis using First DNA Strand as a Template.
This step is carried out by a DNA-dependent DNA polymerase from Escherichia
coli. The first report of cDNA cloning was published in 1970s which was based
on homopolymer tailing technique. The most popular method involved the use of
oligo-dT primer annealing at the polyadenylate tail of the mRNA.
• The oligo-dT primers are used to complete the first strands synthesis. The
primers used are random oligonucleotide hexamers for both the first and
second strand of cDNA synthesis.
• Increasing size of the cDNA renders difficulty in the isolation of full length
clones.
• The reverse transcriptase enzymes used are usually isolated from Avian
Myeloblastosis Virus (AMV) or Moloney Murine Leukemia Virus
(MMLV). The commercially available reverse transcriptase enzymes are
capable of reverse transcriptase activity at temperatures of 50°C.
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 • The native enzymes exhibit optimum activity at 37°C and are affected by Cloning Methodologies

sequences rich in secondary structures. These structures are prevalent in

the 5´ and 3´ untranslated regions.
• The enzyme reverse transcriptase is implied to synthesize the cDNA strands NOTES
from the mRNA. The complementary strand of the mRNA is added with
linker sequences to produce restriction digestion sites.
• The digestion with restriction enzyme leads to formation of cohesive ends in
the cDNA which makes it suitable to be integrated into the vector.
• The specialized enzyme reverse transcriptase converts the single stranded
RNA into complementary DNA.
• At the initial step the first strand is synthesized with an oligo (dT) primer
used for annealing efficiently to the poly(A) tails of the fragment of mRNA.
• The mRNA molecule containing the polyA tail shall be processed for cDNA
preparation.
• The mRNA-cDNA hybrid facilitates in the synthesis of the second strand.
Prior to addition of DNA polymerase, RNAase H is added to remove the
single stranded mRNA-cDNA transcripts. The enzyme RNAase H digests
the RNA strand at specific sites thus creating ssRNA fragments which remain
bound to the ssDNA at specific regions.
• The ssRNA fragments are utilized by the DNA polymerase as primers for
the synthesis of the second strand. This results in the synthesis of double
stranded cDNA molecule.
Synthesis of cDNA Library
The method of cDNA preparation enables to clone and maintain the copies of
cDNA in bacterial vectors. Thus the cDNA library is a representative of the RNA
pool present in the tissue. Unlike genomic libraries, the contents of the cDNA
library depend upon the developmental stage of the tissue. The cDNA library is
likely to contain clones of alternately spliced forms of the mRNA. M13 or M13mp8
vectors are implied to clone cDNAs in abundant levels. Each of the clones are
used for sequencing and identification of proteins encoded. The phage-lambda
vectors are conveniently used for the cloning of cDNA. The lambda gt 10 and
lambda gt 11 and are capable of accepting 7.2 and 7.6 kb size of foreign DNA.
The lambda gt 10 vector is utilized for screening of cDNA by the process of
hybridization. The hybrid vector produced for cDNA cloning possesses both the
features of bacteriophage lambda and plasmids. This type of vector is termed as
the lambda ZAP vectors and possess the following advantages:
• Capable of accommodating up to 10 kb of foreign DNA.
• The presence of a polylinker with specific restriction sites which enables
directional cloning.
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Cloning Methodologies • The presence of T3 and T7 RNA polymerase sites flanking the polylinker
which allows sense and antisense RNA to be prepared from the insert.
These features are included in the plasmid termed as pBluescript and further
NOTES inserted into the phage genome. The further advantage of this vector is that the
cDNA clone can be removed from the phage and multiplied as a high copy number
plasmid.
Construction of cDNA Library
The cDNA is the representative of some percentage of the transcriptome which
are regulative of temporal and spatial expression of the cells. The main advantage
of cloning the cDNA is the absence of introns on the copies of cDNA. Thus, the
non-coding part of the protein is absent in the sequence of cDNAs. This feature
directly facilitates to decipher the sequence of the protein.
Methodology for Construction of the cDNA
The process of construction of cDNA library involves the four major steps which
are mainly as follows,
• Isolation of mRNA .
• Synthesis of the First and the Second Strand of cDNA.
• Integration of cDNA into the Vector.
• Cloning of cDNA to produce the cDNA Library.
Isolation of mRNA: The total mRNA pool pf the desired sample tissue is
preferably extracted by either of the methods like density gradient centrifugation,
chromatographic separation of mRNA using oligo-dT column which retains the
mRNA. Unlike rRNA or tRNAs the mRNA chains contain a 5-250 adenylate
residue poly A tail at its 3´ end. This property allows mRNA to be separated by
oligo-dT matrix in chromatographic column. The other types of RNA molecules
are usually not bound to oligo-dT matrix and usually pass down the column. The
bound form of mRNA is isolated using low-salt buffer.
Synthesis of the First and Second Strand of cDNA: mRNA molecules being
single stranded cannot be used directly in cloning as it is not an ideal substrate for
DNA ligase. Thus, in order to be incorporated into a vector it requires to be
converted to DNA. The activity of reverse transcriptase (RNA-dependent DNA
polymerase) which is obtained from Avian Myeloblastosis Virus (AMV). The
following steps are involved in the synthesis of the first and second strand of cDNA
(Refer Figure 9.6).
• The enzyme reverse transcriptase is implied to synthesize the cDNA strands
from the mRNA. The complementary strand of the mRNA is added with
linker sequences to produce restriction digestion sites.
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• The digestion with restriction enzyme leads to formation of cohesive ends in Cloning Methodologies

the cDNA which makes it suitable to be integrated into the vector.

• The specialized enzyme reverse transcriptase converts the single stranded
RNA into complementary DNA.
NOTES
• At the initial step the first strand is synthesized with an oligo (dT) primer
used for annealing efficiently to the poly(A) tails of the fragment of mRNA.
• The mRNA molecule containing the polyA tail shall be processed for cDNA
preparation.
• The mRNA-cDNA hybrid facilitates in the synthesis of the second strand.
Prior to addition of DNA polymerase, RNAase H is added to remove the
single stranded mRNA-cDNA transcripts. The enzyme RNAase H digests
the RNA strand at specific sites thus creating ssRNA fragments which remain
bound to the ssDNA at specific regions.
• The ssRNA fragments are utilized by the DNA polymerase as primers for
the synthesis of the second strand. This results in the synthesis of double
stranded cDNA molecule.

Fig. 9.6 Method of cDNA Synthesis

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Cloning Methodologies Integration of cDNA into Vector: The ds-cDNA is synthesized from the modified
blunt ended cDNA termini. Blunt end ligation possess inefficiency and short
restriction site linkers are ligated to both the ends. The linker is a double stranded
DNA fragment which possess a particular sequence for restriction enzyme. The
NOTES chemically synthesized oligonucleotides are joined together to form 10-12 base
pair fragments. The blunt ended ds-DNA is ligated to linker DNA which is catalyzed
by DNA ligase obtained from T4 bacteriophage. The cDNA attached with linker
DNA is digested with restriction enzyme which liberates sticky ends. To avoid the
access of restriction sites present in the cDNA fragment the specific bases are
methylated using the enzyme methylase. The internal recognition sequences are
methylated to protect from restriction enzymes. Followed by methylation in the
final step, the ligation of the cDNA into the vector is important to construct a
cDNA library. The plasmid or bacteriophage vectors are digested with the same
restriction enzyme used for linkers. The Escherichia coli cells are transformed
with the recombinant vector (Refer Figure 9.7).

Fig. 9.7 Modification of cDNA Fragments using Linker DNA

Fig. 9.8 DNA Sequence Methylation

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Cloning of cDNA to Produce cDNA Library Cloning Methodologies

The cDNA molecules are cloned inside the phage insertion vectors which provide
certain advantages over the plasmids. These vectors are suitable for the cloning of
higher number of recombinants required for cloning less abundant mRNA molecules. NOTES
The bacteriophage vectors are capable of accommodating a larger number of
phage clones in comparison to plasmid containing bacterial colonies. However,
plasmid vectors are used for cloning during isolation of cDNA sequence associated
with the screening of a relatively short number of clones.
Certain limitations associated with the process of cDNA cloning are mainly
due to longer size of the mRNA fragment. This results in the inefficient synthesis of
full length cDNA. Improper synthesis of cDNA strand causes inefficient expression
of the sequenced gene. The functional efficiency of the reverse transcriptase enzyme
may also regulate the efficiency of cDNA synthesis. Application of S1 nuclease
during cleaving of ds cDNA. This limitation can be overcome by the use of efficient
Escherichia coli vector which can prevent incomplete synthesis of RNA. The use
of S1 nuclease can be substituted by the addition of poly C tail at the 3´ end of the
single stranded cDNA which is copied from the mRNA catalyzed by the activity
of Terminal Deoxynucleotidyl Transferase (TDT). Poly-G oligonucleotide is further
used for the synthesis of complementary strand to yield ds-cDNA.
9.2.5 Cloning from Genomic DNA Library
The genomic DNA library contains most of the gene sequences present in the
genome of an organism. The construction of genomic library is accomplished by
using vectors which are capable of accommodating large sized inserts. The number
of clones required to produce a genomic library largely depends upon the genome
size of the organism and the capacity of the vectors to accommodate the clones.
The selection of host organism (either Prokaryotic or Eukaryotic) is crucial to
obtain success in the process of construction of genome library. The efficiency of
the heterologous expression of protein thus depends upon the selection of host
organism. The main process of construction of a genomic DNA library is
accomplished by its extraction, restriction digestion and cloning into suitable vector.
The recombinant DNA is formed by annealing of the vector DNA (digested with
the same restriction enzyme) along with the fragment of genomic DNA. The
restriction digested genomic DNA fragments are expected to possess more than
one gene.
Isolation and Digestion of Genomic DNA
In the process of DNA extraction, initially the nuclei content are isolated using
protease digestion followed by phenol-chloroform extraction. Usually higher
molecular weight DNA is isolated by CTAB method. The isolated DNA can be
further purified by caesium chloride density gradient centrifugation. The entire
genomic DNA is required to be fragmented by either physical method or by using
restriction enzymes. The time of incubation and concentration of the restriction
enzyme used is essential to obtain success in the fragmentation of the entire DNA.
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Cloning Methodologies In the physical method of DNA fragmentation the DNA is sheared by narrow
gauge syringe and in a sonicator. The average size of genomic DNA should be
preferably around 20 kb in order to be accommodated into a λ based vector.
This method being a random process requires larger quantity of DNA. Furthermore,
NOTES the DNA fragments obtained may be of variable size.
The enzymatic method of DNA digestion possesses a major limitation
regulated by the frequency of restriction sites present in the DNA fragment. This
shall result in DNA fragments of different sizes. The gene of interest required to be
cloned may contain multiple restriction sites present in the DNA. This may, however,
lead to formation of multiple fragments. This problem can be overcome by using
know quantity of enzyme for initial digestion of the genomic DNA, which shall
consequently liberate fragments of ideal size. The choice of the restriction enzyme
depends upon the requirement of blunt or sticky ends produced in the DNA
fragments which are further ligated to the cloning vector. The activity of certain
restriction enzymes can be affected by the modification (methylation) in the DNA
bases. The DNA is digested by restriction enzyme which produces blunt or sticky
ends. Certain enzymes produce blunt ends in the DNA fragments like HaeIII and
AluI. Prior to cloning of these DNA fragments the blunt ends are attached with
linkers (oligonucleotides) which contain sequences for digestion by restriction
enzymes.
Use of Linkers and Adapters
The linkers are short stretches of double stranded DNA fragments of 8-14 bp
(base pair) which possess recognition site for restriction enzymes. The linkers are
ligated to the DNA fragment catalyzed by the enzyme ligase. The attachment of
linker is followed by application of restriction enzyme which produces cohesive
ends in the DNA fragments thus making it suitable for cloning in the vector. The
major limitation in the use of the restriction enzyme is the presence of recognition
site within the DNA fragment desired to be cloned. Conversion of blunt ends of
DNA fragments into cohesive ends can also be accomplished by the use of adapters.
Adapters are short segments of nucleotide molecules which already possess
cohesive ends. These sequences are usually linker segments previously digested
by the restriction enzymes. Certain restriction enzymes like au3AI (recognition
sequence 5´-GATC-3´) are capable of generating DNA fragments that are
complementary to the sticky end produced by BamHI (recognition sequence 5´-
GGATCC-3´).
9.2.6 Cloning of Genomic DNA
The commonly implied vectors necessary for cloning of genomic DNA are namely
λ phage, yeast artificial chromosome, and bacterial artificial chromosome.
λ replacement vectors like λ DASH and EMBL3 are suitable for construction of
genomic DNA library. The λ EMBL series of vectors are implied for constructing
genomic library. The flanking regions of this vector contain opposed promoters
for T3 and T7 polymerases. The restriction digestion of this recombinant vector
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liberates shorter fragments of insert DNA which remain attached to the promoters. Cloning Methodologies
Cosmids, bacterial artificial chromosomes (BACs), P1-derived Artificial
Chromosomes (PACs) and Yeast Artificial Chromosomes (YACs) are some of
the high capacity cloning vectors used for construction of genomic library. These
vectors can accommodate larger size of DNA fragments in comparison with that NOTES
of λ replacement vectors. The recombinant vectors along with insert combinations
are grown in the Escherichia coli cells. Ligation of genomic DNA into the vector
liberates single bacterial colony or a viral plaque. The number of clones required
for constructing an entire genomic library depends upon the size of the genome
and average size of each of the cloned DNA. The number of independent
recombinants present in a genomic library should be essentially higher than that
required. Sampling variations may result in the exclusion or inclusion of the
recombinants from the library. The larger size of the genome library provides higher
chances of obtaining the gene of interest by screening. The initial primary library
created in bacterial or phage colony is usually of low titer. Thus, the primary library
is amplified by means of plating the bacterial or phage colonies. The amplified
library is of larger size and can be stored for longer shelf life associated with phage
colonies. The amplified library obtained from the primary library may be subjected
to some compositional changes. This is attributed to the DNA fragments which
may appear toxic to the growth of Escherichia coli cells in the colony. These DNA
sequences may be lost or under represented in the amplified colonies.
Screening of Genomic DNA Library
The genomic DNA library can be screened for the gene of interest. This process is
accomplished by methods like probe hybridization, western blot method, or by
assay of protein activity. The probe hybridization method involves binding of probe
with the complementary DNA. Figure 9.9 illustrates the flow chart for genomic
library.

Fig. 9.9 Flow Chart for Genomic Library

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Cloning Methodologies Colony Hybridization Method: The method involves hybridization of the probe
with the target DNA fragment. In the initial step of hybridization the colony of host
cells are plated into a solid medium containing screening antibiotics. The transformed
cells containing the antibiotic resistance gene in their plasmid are capable of growing
NOTES in the antibiotic containing medium. The desired discrete colony appearing on the
plate are transferred into the nitrocellulose membrane with its position maintained.
The attached cells are lysed, de-proteinised and the DNA released is denatured in
the presence of alkali. The labelled DNA probe is hybridized to the target DNA
present in the membrane. The presence of gene of interest is determined by
autoradiography which exhibits the presence of spot in the X-ray film. The positional
detection of the colony is followed by the reculture and cloning the gene of interest
(Refer Figure 9.10).

Fig. 9.10 Colony Hybridization Method

Plaque Hybridization: In this method a bacterial culture is initiated on the agar

plate and incubated with a high number of phage particles containing the inserted
DNA fragments. The plaques formed are due to infection of the bacteria with
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phage’s. The plaques are transferred into the nitrocellulose membranes. Radioactive Cloning Methodologies

labelled DNA or RNA probe is applied for hybridisation. The position of the spot
is detected to locate the colony in the main culture.
Screening by Immunological Method: This method is an alternative to the
NOTES
probe mediated detection of gene in a screening. The cells are allowed to transcribe
and translate to express the protein. The proteins are detected by binding with
specific antibody. The discrete colonies are transferred into the nitrocellulose
membrane. The cells are lysed and the proteins are bound to the matrix. The
proteins are incubated with primary antibody followed by secondary antibody.
The secondary antibody is conjugated with enzyme which produces colour in the
presence of substrate. The chromogenic detection indicates the protein present in
the membrane. The immuno-detection of protein confirms the presence of gene of
interest in the colony (Refer Figure 9.11).

Fig. 9.11 Immunological Screening

Chemiluminescence: Chemiluminescence (also chemoluminescence) is the

emission of light (luminescence), as the result of a chemical reaction. The method
of detection by chemoluminescence is accomplished by the activity of the enzyme
horse radish peroxidase and alkaline phosphatase catalyzing the substrate mediated
reaction. The enzymes are tagged to the specific nucleic acid probes.
9.2.7 Shotgun Cloning
The method of shotgun cloning involves fragmentation of genomic DNA by the
application of restriction enzymes. Random physical method can also be used to
cleave DNA into fragments. The DNA in question can be either genomic DNA or
a clone obtained from Yeast Artificial Chromosome (YAC). The larger fragments
of DNA can be inserted into cloning vectors with lower accommodating capacity.
DNA sequencing can be accomplished by shotgun cloning method where the
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Cloning Methodologies sequencing is completed in two or three halves of 600-700 bases in each part
(Refer Figure 9.12).
• The DNA materials are cut into fragments of 160-170 kb. This is
accomplished by restriction enzyme activity or by sonication.
NOTES
• Each fragment is inserted into the Bacterial Artificial Chromosome (BAC)
and multiplied to several copy numbers.
• The 160 kb fragments are further isolated and overlapping 1 kb fragments
are obtained.
• The smaller fragments are further cloned in Escherichia coli cells to obtain
several copies.
• Each of the fragments are now sequenced with the contigs of overlapping
sequences. The set of the overlapping DNA sequence of DNA fragments is
known as a contig.
• The entire DNA fragment of 160 kb is sequenced by assembling across the
overlapping ends.

Fig. 9.12 Method of Shotgun Cloning

Check Your Progress

1. What is cloning?
2. How is a complementation test helpful?
3. What is sticky and blunt end cloning?
4. Give the features of features of blunt end cloning method.
5. What does the process of cDNA synthesis involves?

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 Cloning Methodologies
9.3 ANSWERS TO CHECK YOUR PROGRESS
QUESTIONS

1. Cloning is the process of taking genetic information from one living thing NOTES
and creating identical copies of it.
2. The method of α (alpha) complementation helps in screening of recombinant
plasmids present in the bacterial cells which contain the gene of interest
inserted into the plasmid. This method can distinguish the bacterial colonies
which possess only plasmids while the ones which contain plasmids with
the GOI (Gene Of Interest) integrated in them.
3. Sticky and blunt end cloning of double stranded DNA into the vector appears
to be the most important step in the methodological steps of molecular
biology. The process of gene cloning is associated with sequencing, preparing
DNA libraries and in expression of coding and non-coding RNA.
4. Following are the features of blunt end cloning method:
• This method appears around 10-100 times less efficient than sticky end
method. This inefficiency results due to absence of colonies or wrong
insertion of gene of interest in the recombinant vectors.
• Tendencies of vectors being re-circularized lead to failure in the integration
of GOI. This results in the presence of empty vectors. Thus prior to
sequence verification the vectors should be screened by restriction
digestion or PCR amplification.
• A major advantage of the blunt method is that it does not require any
restriction digestion site in its sequence. Thus, this method excludes any
unwanted sequence additions which might complicate the process. This
method therefore appears to be executed comparatively at a faster rate.
5. The process of cDNA Synthesis involves three major steps namely:
• First Strand DNA Synthesis from the mRNA Strand by the activity of
Reverse Transcriptase.
• Removal of RNA Strand.
• Second Strand DNA Synthesis using First DNA Strand as a Template.

9.4 SUMMARY

• The method of α complementation helps in screening of recombinant

plasmids present in the bacterial cells which contain the Gene Of Interest
(GOI) inserted into the plasmid.
• The α complementation method can distinguish the bacterial colonies which
possess only plasmids while the ones which contain plasmids with the GOI
integrated in them. Self-Instructional
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Cloning Methodologies • The method is based on the assay of an enzyme called β-galactosidase
acting in the substrate X Gal (5-bromo-4-chloro-indolyl-β-D-
galactopyranoside).
NOTES • The recombinant cells contain vectors integrated with the gene of interest
will appear white in nature while the non-transformed colonies will appear
blue in colour.
• X Gal is a widely used substrate used for assaying the activity of the enzyme
β-galactosidase.
• The assay of the active form of β-galactosidase enzyme is possible by
using X Gal in an agar plate.
• The catalytic activity of β-galactosidase enzyme cleaves X Gal to produce
5-bromo-4-chloro-indoxy which upon dimerization and oxidation leads to
formation of a blue coloured insoluble compound called 5, 5´-dibromo-4,
4´-dichloro-indigo.
• Cloning of double stranded DNA into the vector appears to be the most
important step in the methodological steps of molecular biology.
• The process of gene cloning is associated with sequencing, preparing DNA
libraries and in expression of coding and non-coding RNA.
• The blunt ends of the vectors (if any) produced by restriction digestion are
required to be dephosphorylated. This process is accomplished by the
application of alkaline phosphatase enzyme. This results in the prevention
of relegation of vector without addition of the insert.
• The blunt end method is executed for integration of dsDNA into the linearized
plasmid vector where both the fragments possess blunt ends without any
overhanging termini at their ends.
• cDNA molecules represent the mRNA population of a tissue and represents
the diversity of the spliced isoforms of mRNA.
• The cellular mRNA is reverse transcribed to form the cDNA fragment.
• The process of cDNA synthesis involves three major steps namely, (a)
First strand DNA synthesis from the mRNA strand by the activity of reverse
transcriptase, (b) Removal of RNA strand, and (c) second strand DNA
synthesis using first DNA strand as a template.
• The genomic DNA library contains most of the gene sequences present in
the genome of an organism.
• The construction of genomic library is accomplished by using vectors which
are capable of accommodating large sized inserts.
• The method of shotgun cloning involves fragmentation of genomic DNA by
the application of restriction enzymes.

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 Cloning Methodologies
9.5 KEY WORDS

• Cloning: Cloning is the process of taking genetic information from one living
thing and creating identical copies of it. NOTES
• Chemiluminescence: Chemiluminescence, also chemoluminescence is the
emission of light, i.e., luminescence, as the result of a chemical reaction.
• Shotgun cloning: The method of shotgun cloning involves fragmentation of
genomic DNA by the application of restriction enzymes.

9.6 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is cloning? Name the various methods of cloning.
2. Define the significance of α complementation test.
3. How is detection of transformants done by complementation method?
4. What is sticky and blunt end cloning?
5. How is cloning from genomic DNA library done?
6. Define the significance of shotgun cloning.
Long Answer Questions
1. Discuss the various methods of cloning.
2. Explain briefly the principle and significance of α complementation method.
3. Elaborate on the synthesis of cDNA giving appropriate examples.
4. Explain the procedure of cDNA cloning. What are the advantages of a
cDNA library?
5. Explain the methodology involved in cloning from genomic DNA.
6. Describe the procedure of genomic DNA library preparation.
7. Explain the procedure of shotgun cloning method.

9.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers. Self-Instructional
Material 197
Cloning Methodologies Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
NOTES Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 Screening of

UNIT 10 SCREENING OF Recombinants, Blotting

Techniques and Blotting
Techniques
RECOMBINANTS,
NOTES
BLOTTING TECHNIQUES
AND BLOTTING
TECHNIQUES
Structure
10.0 Introduction
10.1 Objectives
10.2 Screening of Recombinants: Phenotypic Expression of Characters
10.3 Blotting Techniques
10.4 Mapping of Human Genes: Human Genome Project
10.5 Answers to Check Your Progress Questions
10.6 Summary
10.7 Key Words
10.8 Self Assessment Questions and Exercises
10.9 Further Readings

10.0 INTRODUCTION

Screening for recombinants is one of the most crucial and time-consuming steps in
molecular cloning and several approaches available for this purpose include colony
PCR screening, blue white screening, screening of recombinants, which have the
gene of interest in the MCS region of the cloning vehicle, in such a way that the toxic
gene reading frame is interrupted making the toxic gene inactivated upon insertion
of any foreign gene; GFP fluorescence vectors wherein upon cloning, the GFP
fluorescence disappears, etc. The method for screening of bacterial transformants
that carry recombinant plasmid with the gene of interest, has become more rapid
and simple by the use of vectors with visually detectable reporter genes.
Biochemistry studies molecules, such as DNA, RNA and proteins. Blotting
techniques are what scientists use to separate these types of molecules. In cells,
they exist as a mixture. Blotting allows researchers to find one protein among
many, like a needle in a haystack. Blotting is generally done by letting a mixture of
DNA, RNA or protein flow through a slab of gel. This gel allows small molecules
to move faster than bigger ones. The separated molecules are then pressed against
a membrane, which helps move the molecules from the gel onto the membrane.
The molecules stick to the membrane, but stay in the same location, apart from
each other, as if they were still in the gel. Genetic mapping offers evidence that a
disease transmitted from parent to child is linked to one or more genes and provides
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Screening of clues about which chromosome contains the gene and precisely where the gene
Recombinants, Blotting
Techniques and Blotting lies on that chromosome. Among the main goals of the Human Genome Project
Techniques (HGP) was to develop new, better and cheaper tools to identify new genes and to
understand their function. One of these tools is genetic mapping. Genetic mapping
NOTES - also called linkage mapping - can offer firm evidence that a disease transmitted
from parent to child is linked to one or more genes. Mapping also provides clues
about which chromosome contains the gene and precisely where the gene lies on
that chromosome.
Genetic maps have been used successfully to find the gene responsible for
relatively rare, single-gene inherited disorders, such as Cystic Fibrosis (CF) and
Duchenne muscular dystrophy. Genetic maps are also useful in guiding scientists
to the many genes that are believed to play a role in the development of more
common disorders such as asthma, heart disease, diabetes, cancer, and psychiatric
conditions.
In this unit, you will study about screening of recombinant, phenotypic
expression of characters, blotting techniques - western, northern and southern,
mapping of human genes, human genome project in detail.

10.1 OBJECTIVES

After going through this unit, you will be able to:

• Discuss the various methods of screening of recombinant clones
• Understand what blotting technique is
• Explain about the mapping of human genes

10.2 SCREENING OF RECOMBINANTS:

PHENOTYPIC EXPRESSION OF CHARACTERS

The process of screening enables to find the gene of interest present in the cloning
vector of a particular colony. The recombinant vectors containing the DNA fragment
along with marker antibiotic gene are suitable to be grown in antibiotic supplemented
medium. Bacterial and phage colonies appear to exhibit definite plaqued
appearance which is further screened by probe hybridization or immunological
screening. The mechanisms of some of the methods of screening of recombinants
are mainly based on changes in phenotypic expression of characters. Cloning
methods are successfully accomplished after the detection of the gene of interest.
Thus gene screening requires the correct identification of the transformed cells
which bear the gene of interest being inserted into the cloning vector. Largely the
process of cloning and obtaining the recombinants involve methods like direct
selection or clone identification from a gene library. In the direct selection method
the clone obtained contain the required gene inserts. This conventional method is
faster does not usually obtain false positive results. In the process of selection of
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clone from a colony a random physical method can also be used to cleave DNA Screening of
Recombinants, Blotting
into fragments. The DNA in question can be either genomic DNA or a clone Techniques and Blotting
obtained from Yeast Artificial Chromosome (YAC). The larger fragments of DNA Techniques
can be inserted into cloning vectors with lower accommodating capacity. DNA
sequencing can be accomplished by shot gun cloning method where the sequencing NOTES
is completed in two or three halves of 600-700 bases in each part. This process
proceeds in the form of a shot gun cloning which results in identification.
Methods of Direct Screening of Clone
Following are the methods of direct screening of clone.
Antibiotic Resistance Screening
The method utilizes the principle of identifying the transformed cells by means of
antibiotic selection. The DNA of the foreign gene is cloned within the frame of a
coding gene responsible for a particular phenotype. Insertion of the foreign gene
results in the disruption of the gene product thus causing changes in the phenotypic
expression in the host. The insertion inactivation method can be successfully implied
on plasmids containing antibiotic resistance genes. The pBR322 plasmid contains
two antibiotic resistance genes for Ampicillin and Tetracycline. The specific region
of the plasmid known as ScaI accommodates the DNA fragment of the foreign
gene. Insertion of the gene in ScaI region results in Ampicillin sensitivity and
tetracycline resistance in the recombinant plasmid. The screening of recombinant
plasmids are accomplished by plating the recombinant Escherichia coli cells in
Tetracycline supplemented medium. The replica plate is grown separately on
ampicillin supplemented medium. Plating in two different media will result in
comparison of the colonies unable to grow in ampicillin medium. Figure 10.1
illustrates the method of antibiotic resistance gene inactivation method.

Fig 10.1 Method of Antibiotic Resistance Gene Inactivation Method

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Screening of Blue White Screening Method
Recombinants, Blotting
Techniques and Blotting The method is based on the assay of an enzyme called b-Galactosidase acting in
Techniques
the substrate X Gal (5-bromo-4-chloro-indolyl-β-D-Galactopyranoside). Due
NOTES to reasons of the differences appearing in the colour of colonies, the method is
known as blue-white screening method. Thus, success of ligation in vector-based
cloning methods can be confirmed by this complementation test. The DNA of
interest is integrated into the vector and transformed into the Escherichia coli
cells for cloning. The transformed cells of the bacteria are preferentially grown in
the presence of X Gal in the growth medium. The recombinant cells contain vectors
integrated with the gene of interest will appear white in nature while the non-
transformed colonies will appear blue in colour. X Gal is a widely used substrate
used for assaying the activity of the enzyme β-Galactosidase. X Gal is chemically
an Indoxyl Glycoside Ester molecule which upon hydrolysis by the enzyme
produces a blue compound. The β-Galactosidase enzyme is structurally a
homotetramer in its active state which is encoded by the lac Z gene of the Lac
Operon.
Methods of Gene Clone Selection from a Library
The process of gene clone selection from a library can be performed by nucleic
acid hybridization, functional screening or chromosome walking techniques,
respectively.
Nucleic Acid Hybridization Method
The method of clone detection by hybridization involves complementary binding
between the DNA sequences (GOI - Gene Of Interest) with specific
oligonucleotide probes added in the reaction. Initially the DNA is extracted from
the transformed cells or phage vectors and transformed into a nylon membrane.
The oligonucleotide probe implied should possess complete or partial sequence
similarity with the DNA from the GOI. The DNA is denatured to form single
stranded structure which is immobilized to the membrane and hybridized to the
radioactive probe. Followed by incubation of the membrane in the probe solution
the membrane is washed to remove the excess unbound probe.
Functional Screening
The method of functional screening is associated with the protein analysis (end
product) using an expression vector. The proteins likely to be produced from the
genes are obtained by inserting clones into the expression vector. The immuno-
screening of the proteins confirm the presence of the desired gene in the clone.
The process involves incubation of membrane with antibodies which bind to the
protein by the virtue of epitope specificity. The molecular interactions involve
covalent bond formation between the protein and antibodies.
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Chromosome Walking Screening of
Recombinants, Blotting
The fragments of genomic DNA clones are separately screened with use of probes Techniques and Blotting
Techniques
necessary for detecting the gene of interest. The clones are screened for the GOI
by hybridizing with the radioactive probes. Thus the method of chromosome walking
NOTES
involves screening of the ends of overlapping clones with the relevant probe or
DNA segment.

Check Your Progress

1. How is process of screening helpful?
2. Define direct selection method.
3. What is insertion inactivation method?

10.3 BLOTTING TECHNIQUES

A blot, in molecular biology and genetics, is a method of transferring proteins,

DNA or RNA, onto a carrier (for example, a nitrocellulose, Poly VinyliDene
Fluoride (PVDF) or Nylon membrane). In many instances, this is done after a gel
electrophoresis, transferring the molecules from the gel onto the blotting membrane,
and other times adding the samples directly onto the membrane. After the blotting,
the transferred proteins, DNA or RNA are then visualized by colorant staining (for
example, silver staining of proteins), autoradiography visualization of radioactive
labelled molecules (performed before the blot), or specific labelling of some proteins
or nucleic acids. The latter is done with antibodies or hybridization probes that bind
only to some molecules of the blot and have an enzyme joined to them. After proper
washing, this enzymatic activity (and so, the molecules we search in the blot) is
visualized by incubation with proper reactive, rendering either a coloured deposit
on the blot or a chemiluminiscent reaction which is registered by photographic film.
Western Blot Method
The method is applied for immunological detection of protein molecules earlier
separated by PolyAcrylamide Gel Electrophoresis (PAGE). This method enables
to detect a particular protein from a mixture of proteins. The major steps of this
method involve:
• Separation of protein molecules on the basis of mass of the polypeptides.
• Transfer of the proteins into the nitrocellulose or PVDF membrane (blot
transfer).
• Immunological detection of the proteins using primary and secondary
antibodies.
The technique has been named by the scientist called W. Neal Burnette.
The method of western blot involves certain variations and modifications associated
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Screening of with sample preparation, blot transfer and immuno-staining. The success of the
Recombinants, Blotting
Techniques and Blotting method depends upon the specific protocol used for protein extraction and
Techniques separation on PAGE. The protocol for protein extraction depends upon the type
of analytical sample or tissue used for protein detection. The method determines
NOTES the expression of protein isoforms and its quantitative expression in various sample
tissues. The PAGE technique implied for separation of proteins during western
blot should essentially be a SDS-PAGE method.
Methodology for Western Blot
Sample Preparation for Protein Homogenate
The process of protein sample preparation includes selection of correct lysis buffer
functioning in a proper range of pH. Separation of membrane proteins requires
the application of strong denaturants like Dithioerythritol, Sodium Dodecyl Sulphate
or CHAPS buffer. The extraction of cytosolic proteins can be accomplished by
using Sodium Phosphate or Tris-HCl buffer. The Lysis buffers should preferably
contain protease inhibitors which shall prevent the process of protein degradation.
beta-Mercaptoethanol, or DTT is used in the Lysis buffer which cleave the sulphide
bonds present between the cysteine residues of the protein subunits. Glycerol is
commonly used in the Laemmli buffer which acts as an Osmoticum and allows the
samples to sink in the wells of the stacking gel (Refer Figure 10.2).

Fig. 10.2 Methodology for Western Blot

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Sample Preparation for Total Cytosolic Protein Extraction Screening of
Recombinants, Blotting
Plant samples (500 mg/1gm) are ground to powder in liquid nitrogen and the powder Techniques and Blotting
Techniques
is transferred into Eppendorf tubes and incubated in the homogenization buffer (25
mM Tris-HCl, 400 mM Sucrose, 10 mM KCl, 1mM MgCl2, 1mM EDTA-Disodium
NOTES
Salt, 1 mM PMSF, 0.2% β-Mercaptoethanol, pH 7.5) at a proportion of 1.2 ml
g-1 FW for 30 minutes at 4°C in a vortex shaker. The homogenates are centrifuged
at 12,000 g for 20 minutes at 4°C and supernatants collected are further centrifuged
at 10,000 g for 20 minutes at 4°C to pellet fine suspended particles.
Sample Preparation for Membrane Proteins
Samples are homogenized in Tris-Urea buffer (50 mM Tris, pH 7.5, containing 9
M Urea and filtered through 4 layers of muslin cloth. The filtrate thus obtained is
centrifuged at 10,000 g at 4°C for 20 minutes. The oil body pads collected is
subsequently resuspended in 1.25 mL NaHCO3 (0.1 M) per gram of tissue and
incubated at 4°C for 30 minutes and again centrifuged at 10,000 g for 20 minutes
at 4°C. The oil body pads are then resuspended in excess Tris-Sucrose buffer
(20 mM Tris, 0.2 M Sucrose, pH-7.5) to wash out excess bicarbonate. After
centrifugation at 10,000 g for 20 minutes at 4°C, the oil body pads are resuspended
in the same buffer. In order to solubilize oil body membrane proteins, oil body
suspension washed in Tris-Sucrose buffer is extracted four times with five volumes
of Diethyl Ether. Residual Diethyl Ether is evaporated from each sample at room
temperature while vortexing continuously. The residues containing oil body
membrane proteins is suspended in Tris-Sucrose buffer. 160 µL of this suspension
is mixed with 40 µL of 10% SDS solution. The samples are heated at 90 °C in a
water bath for 30 min in order to solubilise membrane proteins, and centrifuged for
15 minutes at 7000 g and 4°C. Oil body membrane proteins in the supernatant are
quantified according to Markwell’s method of protein quantification.
Concentrating of Protein Aliquots
Protein aliquots obtained may have variable protein content (µg/mL) depending
upon the age and type of plant tissues used. However gel electrophoresis requires
good amount of protein content (25-100 µg) required to be loaded in
electrophoresis gels having limitations in the volume of wells which vary from
(30-50 mL). Thus, to reduce water content and volume of protein aliquots,
supernatants obtained are concentrated using Vivaspin 2 kDa MWCO columns
(GE Health Care, UK) at 10,000g. Prior to concentrating, the columns are saturated
with homogenisation buffer for 15 minutes at 10,000 g to avoid drying of membrane.
Purification of Protein Samples
Protein homogenates after concentrating require purification steps in order to reduce
phenolic and Lipidic constituents. These impurities if present often interfere with
various stains used in protein staining gels. Phenolic compounds along with degraded
DNA or RNA products often cause streaking and intense background stains in
the electrophoretograms.
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Screening of Delipidation Steps
Recombinants, Blotting
Techniques and Blotting
Techniques
Concentrated homogenates obtained are proceeded for delipidation steps by
acetone washings. Homogenates are incubated with 100% Acetone (1:3 v/v)
NOTES overnight at -20°C followed by gentle vortex and centrifugation at 10,000 g at
4°C for 20 minutes. Supernatants containing all organic contaminants mixed with
Acetone are discarded and the Protein pellets obtained are suspended in 90%
Acetone followed by incubation at -20°C for 20 minutes. After incubation pellets
are collected by centrifugation (10,000 g for 20 minutes at 4°C) followed by two
more acetone (90%) washings. Pellets obtained after acetone washings are then
air dried to remove all traces of acetone vapours and suspended in 200 µL
homogenisation buffer. Protein suspensions are then sonicated for 5 minutes and
centrifuged at 10,000 g for 20 minutes at 4°C. Alternatively TCA and chloroform/
methanol mixture can also be used to precipitate proteins and dissolve Lipidic
constituents.
Desalting by Dialysis
To remove salt contaminants, protein samples are dialyzed in dialysis kits overnight
in a dialysis buffer.
Protein Content Estimation by Bradford’s Method
Bradford’s method of protein quantification is one of the most widely used simple
protocols for calculating protein concentrations in terms of µg/mL in sample aliquots.
Denaturants like urea used in protein solubilisation buffer (applicable for extraction
of membrane proteins) often interfere with binding to the dye and liberating false
results. Thus alternatively Lowry’s method or Markwells’s method of protein
estimation can also be used to analyze protein content. Bradford’s method involves
binding of the Comasiie Brilliant Blue dye with proteins which results in a blue
colour complex formation which is detected for absorbance value at 595 nm in a
spectrophotometer. This step should be performed after all protein purification
steps have been performed with the sample aliquots. Standard curve of Bradford’s
estimation involves Bovine Serum Albumin (BSA) solution dissolved in 0.75 M
NaCl solution at a concentration of 1µg/ml. Further dilutions (100, 80, 60, 40 and
20 µg/mL) are prepared with NaCl solutions. Standard and sample aliquots are
incubated separately with Bradford’s reagent in dark at 25ºC for 20 minutes
followed by which absorbance is recorded at 595 nm. Blank containing only
Bradford’s reagent and homogenisation buffer is used as a control to set reference
or zero in spectrophotometer The standard curve plotted with absorbance and
concentration parameters is used to derive the value of 0.1 absorbance value
equivalent to the amount of protein in mg. The absorbance thus obtained from
samples is then calculated for amount of protein present.
Calculations:
0.1 Absorbance = X µg Protein (from Standard Curve of BSA)
Samples with specific absorbance values are calculated for protein content in
respect to the above relation.
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Protein Suspension in Laemmli Buffer Screening of
Recombinants, Blotting
Protein aliquots equivalent to 10 µg of protein are boiled with Laemmli Buffer Techniques and Blotting
Techniques
(1:1, v/v) at 90°C for 2 minutes followed by boiling in 0.2% β-Mercaptoethanol
at 90°C for 2 minutes.
NOTES
Separation of Proteins by SDS-PAGE
Preparation of 12.5 % SDS-Gel
Stock solutions for SDS-PAGE analysis, according to Laemmli (1970)
• Acrylamide stock (30%) was prepared by dissolving 29.2 g of Acrylamide
and 0.8 g Bis-Acrylamide in water to make a final volume of 100 mL.
• Resolving gel buffer (3M Tris buffer, pH 8.8) was prepared by dissolving
36.35 g of Tris base in water. The pH of solution was set at 8.8 with HCl
and final volume was made to 100 mL using distilled water.
• Stacking gel buffer (0.5 M Tris buffer, pH 6.8) was prepared by dissolving
6.06 g of Tris base in water. The pH of solution is set at 6.8 with HCl and
final volume was made to 100 mL using distilled water.
• Sodium Dodecyl Sulphate (SDS) solution (10%; w/v) was prepared by
dissolving 10 g of SDS in water to make a final volume of 100 mL.
• Ammonium PerSulphate (APS) solution (10%; w/v) was prepared by
dissolving 100 mg APS in water to make a final volume of 1 mL.
• TEMED (N, N, N´, N´-TetraMethylEthyleneDiamine) was used undiluted.
• Electrode buffer [0.025 M Tris, 0.192 M Glycine, 0.1% (w/v) SDS] was
prepared by dissolving 15.15 g Tris base, 72 g Glycine and 5 g SDS in
water to make a final volume of 5 L.
All stock solutions were stored at 4°C for long term usage and Ammonium
PerSulphate (APS) was dissolved fresh in water prior to use. SDS solution was
stored at room temperature.
Preparation of Resolving SDS Gel (12.5%): Resolving gel was casted using
the mixture of following components aliquoted from their respective stock solutions
and mixed at room temperature. The composition of the resolving gel mixture is as
follows:
Components Quantity (12.5% Gel)
Acrylamide Stock (30%) 2.5 mL
Resolving Gel Buffer (3M Tris, pH 8.8) 1.5mL
SDS Solution (10%; w/v) 120 µL
TEMED (Tetramethylethylenediamine) 6 µL
Ammonium Persulphate (10%; w/v)
*
120 µL
Water 2.62 mL
*
Freshly prepared and added at last to start polymerization.
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Screening of Preparation of Stacking SDS Gel (5%): Stacking gel was casted using the
Recombinants, Blotting
Techniques and Blotting mixture of following components aliquoted from their respective stock solutions
Techniques and mixed at room temperature. The composition of the stacking gel mixture is as
follows:
NOTES
Components Quantity (5% Gel)
Acrylamide Stock (30%) 400 µL
Stacking Gel Buffer (0.5 M Tris, pH 6.8) 0.5 mL
SDS Solution (10%; w/v) 20 µL
TEMED (Tetramethylethylenediamine) 4 µL
Ammonium Persulphate (10%; w/v)
*
13 µL
Water 1.08 mL
*
Freshly prepared and added at last to start polymerization.

Separation of Proteins using SDS-PAGE: 20 µg protein from each sample

were loaded for single dimension separation on a 12.5 % linear SDS-PAGE in
Miniprotean Tetra Cell (BioRad, U.S.A). The gel was run with step wise increasing
voltage (25V, 35V, 55V and 75 V for 10 minutes each) followed by a constant
voltage of 200V for 100 minutes.
Preparation of Resolving Gradient SDS-PAGE Gel: Resolving gradient gel
(10 to 20%) was prepared by mixing the resolving gel mixtures (10% and 20%)
using a gradient mixer. The composition of resolving gel mixtures is shown in the
following table.

Components Concentrations of Resolving Gel Mixture (%)

10 20
Acrylamide Stock (40% ) 3.8 mL 7.6 mL
Resolving Gel Buffer (3 M Tris, pH 8.8) 1.9 mL 1.9 mL
SDS Solution (10%; w/v) 0.15 mL 0.15 mL
Urea (a), Solid 5.41 g 5.41 g
TEMED (Tetramethylethylenediamine) 10 mL 10 mL
Ammonium Persulphate (b) (10%; w/v) 0.1 mL 0.1 mL
Water (c)
As Required As Required
(a)
Used only for making urea containing gel for the analysis of oil body membrane protein.
(b)
Freshly prepared and added at the end to start polymerization.
(c)
As required to bring the final volume to 15 mL.

Preparation of Stacking Gel: Stacking gel is prepared after polymerization of

the resolving gel. Composition of stacking gel is as follows:

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 Components Quantity Screening of
Recombinants, Blotting
Acrylamide Stock (40% T) 1.9 mL Techniques and Blotting
Techniques
Stacking Gel Buffer (0.5 M Tris, pH 6.8) 1.9 mL
SDS Solution (10%; w/v) 0.15 mL NOTES
Urea (a), Solid 5.41 g
TEMED (Tetramethylethylenediamine) 10 µL
Ammonium Persulphate (b) (10%; w/v) 0.1 mL
Water (c)
As Required
(a)
Used only for making urea containing gel.
(b)
Freshly prepared and added to start polymerization.
(c)
As required to bring the final volume to 15 mL.
The samples of protein homogenates are boiled in laemmli buffer by mixing with
β-Mercaptoethanol. Followed by boiling, the samples are separated by PAGE at
the required voltage and current.
Blot Transfer of Proteins from Gel to Membrane
The process of protein transfer from the gel to membrane is accomplished in a blot
transfer unit which is also monitored at a particular voltage and current. The time
span for transfer depends upon the percentage of the gel and the size of the
polypeptides desired to be transferred. The proteins can be transferred through
both semi-dry and wet method. In the wet method of transfer the assembly of gel
and membrane are sandwiched with filter paper and sponges. The entire assembly
is placed within a tank containing transfer buffer. In the protocol for dry transfer the
assembly is placed directly in the transfer unit without being immersed in the buffer.
• The gels are removed from electrophoresis unit and washed in 40 % methanol
followed by rinsing in distilled water and transfer buffer (25 mM Tris, 192
mM Glycine pH .3, 20% Methanol) for 2 minutes. Six layers of filter paper
are cut according to the size of the Gel and Poly VinyliDene Fluoride (PDVF)
/Nitrocellulose membrane.
• The filter paper, membrane and sponges are pre-soaked in transfer buffer.
• The sandwich is prepared in the order of sponge-filter papers (3)-gel-
membrane-filter paper (3)-sponge.
• The transfer unit is placed within an ice box before connecting to the power
supply.
• The transfer is executed for 90 minutes at 100 volts.
Blocking and Antibody Staining
The process of immuno-staining is important for obtaining proper results of protein
detection. The crucial steps associated with the process involve percentage and
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Screening of time of incubation of blocking solution and the dilution of primary and secondary
Recombinants, Blotting
Techniques and Blotting antibody used for detection.
Techniques
• Blocking is executed by adding 5% BSA (Bovine Serum Albumin) dissolved
NOTES in incubation buffer (PBS). The membranes can be kept at 4°C overnight in
blocking solution or alternatively for 2 hours at low temperature. Overnight
blocking can be preferred to prevent background staining and non-specific
binding of antibodies.
• Followed by blocking the membranes are incubated in the solution for primary
antibody at dilutions of 1:100 or 1:200 dissolved in PBS. Overnight
incubation is preferred for better binding of antibody to the proteins.
• Secondary antibody incubation (1 hour) is performed after washing the
membrane in PBS. The dilution used is in the range of 1:500 or 1:1000.
• The chromogenic detection is accomplished by HRP (Horse Radish
Peroxidase) activity acting on the substrate bound with the secondary
antibody.
Southern Blot Method
This method combines the process of DNA separation by Agarose Gel
Electrophoresis (AGE) followed by detection of gene of interest by using a DNA
probe. In this process the DNA molecules are separated by AGE on the basis of
their mass. The DNA are transferred onto a membrane and immobilized before
hybridization with the fluorescent probe. The process of elution of DNA from the
gel and designing of the probe are important components of the method. In
comparison with other techniques southern blot hybridization offers better resolution
in terms of DNA detection. The method has been after the scientist Edwin southern
who published the applications of the technique in the year 1975. The main
components of the method involve extraction of DNA, digestion of DNA by
restriction enzymes, separation by agarose gel electrophoresis followed by transfer
of DNA into the nitrocellulose membrane.
Methodology for Southern Blot Hybridization
DNA Purification/Extraction
Sample Preparation using CTAB Method: 500 mg of plant sample crushed in
2 mL CTAB buffer. The extract is immersed in water bath at 60°C for 20 minutes.
The extract is centrifuged at 13,000 rpm for 5 minutes at 4°C. Supernatant is
transferred to Eppendorf tubes and equal volume of Phenol : Chloroform : Isoamyl
Alcohol is added in the ratio of (24:24:1; v/v). The mixture is inverted several
times gently. Followed by mixing the aqueous layer is transferred separately and
kept overnight at 20°C by adding absolute alcohol. Followed by overnight
incubation the mixture is centrifuged at 13,000 rpm for 3 minutes. Ethanol is
discarded and DNA pellet is air dried (Refer Figure 10.3).
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 Screening of
Recombinants, Blotting
Techniques and Blotting
Techniques

NOTES

Fig. 10.3 Methodology for Southern Blot

Sample Preparation using Modified CTAB Method: Briefly 500 mg of plant

tissue is ground to powder in liquid nitrogen and transferred into Eppendorf tubes.
The powder is mixed with 300 L EBA, 900 mL EBB, and 100 mL SDS and
vortexed thoroughly. The mixture is incubated at 65°C for ten minutes. The tube is
placed on ice and 410 mL of cold potassium acetate is added and mixed by
inversion. The mixture is further incubated ice for 3 minutes. Followed by incubation
the mixture is centrifuge at 13,000 rpm for 15 minutes at 4°C. Supernatant is
transferred Eppendorf tube and 540 L of ice cold absolute isopropanol is added
followed by incubation in ice for 20 minutes. After incubation centrifugation is
performed at 10,200 rpm for 10 minutes and the supernatant is discarded. The
pellet is washed once in 500 mL 70% Ethanol and allowed to dry. The pellet is
resuspended in 600 mL of TE, 60 mL 3M Sodium Acetate (pH 5.2) and 360 mL
ice cold absolute Isopropanol. The mixture is incubated on ice for 20 minutes.
Reagents and Buffers
Extraction Buffer A (EBA) Per 100 mL
2% (w/v) Hexadecyltrimethylammonium Bromide (CTAB) 2.0 g
100 mM Tris (pH 8.0) (Use 1 M Stock) 10 mL
20 mM EDTA (Use 0.5 M Stock) 1 mL
1.4 M NaCl 8.2 g
4% (w/v) Polyvinylpyrrolidone (PVP) 4.0 g
0.1% (w/v) Ascorbic Acid 0.1 g
10 mM β-Mercaptoethanol (BME)* (Use 14.3 M Stock) 70 mL
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Screening of Extraction Buffer B (EBB) Per 100 mL
Recombinants, Blotting
Techniques and Blotting 100 mM Tris-HCl (pH 8.0) (Use 1 M Stock) 10 mL
Techniques
50 mM EDTA (Use 0.5 M Stock) 2.5 mL
NOTES 100 mM NaCl 0.6 g
10 mM β-Mercaptoethanol (BME)* (Use 14.3 M Stock) 70 mL
TE Buffer Per 100 mL
10 mM Tris (pH 8.0) (Use 1 M Stock) 1.0 mL
1 mM EDTA (Use 0.5 M Stock) 50 µL
Other Required Reagents
20% (w/v) Sodium Dodecyl Sulphate (SDS)
5 M Potassium Acetate (Stored at –20ºC)
3 M Sodium Acetate (pH 5.2)
70% Ethanol (Stored at -20ºC)
Absolute Isopropanol (Stored at -20ºC)
Estimation of DNA
Qualitative Estimation of DNA (Diphenylamine test) Principle: DNA
containing pentose deoxyribose is treated with diphenylamine under acidic
conditions, resulting in formation of a blue compound exhibiting absorbance maxima
at 595 nm. In acid solution deoxypentose is converted to the highly reactive b-
Hydroxy Lerulin Aldehyde, which reacts with diphenylamine to produce a blue
complex. In DNA the deoxyribose of purine nucleotide only reacts to form the
blue complex. Thus the value obtained represents one half of the total deoxyribose
produced.
Stock Standard Solution: 50 mg of DNA is dissolved in 50 mL of saline sodium
citrate buffer (1mg/mL). Diphenylamine Reagent: 10g of pure diphenylamine is
dissolved fresh with 25 mL of concentration sulphuric acid and final volume made
up to 500 mL with glacial acidic. Buffered saline (pH 7.4): Sodium chloride (0.14N)
and sodium citrate (0.02M). Procedure: Working standard solution (DNA) dilutions
are prepared at a range of 50-250 µg/mL in buffer saline. The tubes containing
unknown sample are taken in quantity of 1-2 mL. The volume in all test tubes is
increased to 3 mL with distilled water. Diphenylamine reagent (4 mL) is added to
all the tubes and kept in a boiling water bath at 36°C for 20 minutes. This results
in formation of a blue coloured complex. Absorbance is recorded at 595 nm.
Amount of DNA present is quantified by standard curve plotted with concentration
of DNA on X-axis versus absorbance in Y-axis.
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DNA Fragmentation by Restriction Enzymes Screening of
Recombinants, Blotting
Techniques and Blotting
The required amount of DNA is mixed with the required restriction enzymes. The Techniques
components are added into the PCR tubes. The concentration of the restriction
enzymes used and the time of incubation are important for obtaining proper digestion NOTES
of DNA fragments. The digestion is preferably carried out at a temperature of
37°C by immersing in a water bath. Prolonged digestion for genomic DNA is
accomplished in overnight duration by adding the restriction enzyme in two
instalments. Followed by incubation the DNA sample is mixed with 50 µL TE and
tracking dye to perform Agarose Gel Electrophoresis.
DNA Separation by Agarose Gel Electrophoresis
Agarose gel pore size is suitable for separation of DNA varing in range (0.7 kb to
25 kb). Agarose is a linear polymer of D and L galactose residues alternately
joined by glycosidic linkage. The pores in between the agarose gel matrix helps in
efficiently sieving the DNA molecules. The molecular size of DNA and the
percentage of agarose (pore size) are the prime factors regulating migration rate of
DNA in agarose gel electrophoresis. However, the form of DNA (super helical,
nicked circular or linear) also affects the rate of migration. The voltage applied
along with the ionic strength of electrophoresis buffer affects DNA mobility.
Procedure
The electrophoresis gel casting tray is properly sealed with tapes. The size is adjusted
as per requirement. Sufficient electrophoresis buffer (1X TAE or 0.5X TBE) is
prepared to dissolve agarose and further use in electrophoresis tank. Agarose is
dissolved in electrophoresis buffer to attain a percentage of 0.8% (W/V) and
heated in a microwave. The strength of agarose solution, however, depends upon
the size of DNA intended to be separated. Insulated gloves should be worn and
care should be taken to handle molten agarose. An appropriate comb is selected
(0.5-1.0 mm) and placed at the center of the tray so that wells form as the agarose
solution cools down. The molten agarose solution is gently poured in the gel casting
tray, avoiding bubble formation. The gel is allowed to completely solidify for 45
minutes followed by solidification of gel (3 to 5 mm) electrophoresis buffer is
added on top and combs are gently removed. DNA is mixed with 0.20 times of
6X gel loading buffer and loaded carefully in the wells. 500 ng of DNA is sufficient
to be detected by Ethidium Bromide or SYBR gold stain. The wells should be
loaded with DNA samples without contamination or mixing of samples from
neighbouring wells. Electrophoresis tank should be filled with buffer and completely
immersing the gel. Lids should be covered and required voltage of 1-5V/cm is
applied to run the gel. Gel is allowed to run until tracking dye (bromophenol blue)
migrates to a sufficient distance from the wells.
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Screening of DNA Fragmentation and Blot Transfer
Recombinants, Blotting
Techniques and Blotting
Techniques The process of hybridization requires binding of DNA probe to the single stranded
DNA molecule. Thus, to obtain DNA fragmentation the DNA molecules are
NOTES digested by alkali solutions. Blot transfer involves movement of the fragmented
DNA molecules into the nitrocellulose membrane. Nitrocellulose membranes can
also be replaced by nylon membranes which often exhibit better affinity to DNA
molecules during the process of transfer.
• Followed by agarose gel electrophoresis for a particular duration, the gel is
transferred into the UV mediated gel documentation unit and the bands are
visualized at 36 nm.
• After documentation, the gel is denatured using 1.5M NaCl and 0.5 M
NaOH for 20 minutes. This process is followed by immersing in buffer
containing 0.5 M Tris HCl (pH 7) and 3 M NaCl for 20 minutes.
• The portion of the gel required to be transferred is cut in the form of a strip
and sized with six layers of filter paper and the transfer membrane. All these
components are dipped into the SSC buffer for 2 minutes.
• The transfer assembly containing the filter paper-gel-membrane-filter paper
complex is aligned within a cassette of glass plates without allowing air
bubbles in between.
• The process of transfer is carried out for duration of 2 hours.
• Followed by transfer, the membrane is removed and the membrane is
immersed in standard Denhardt’s solution for 1 hour.
• The membrane is dipped in pre-hybridization solution containing required
amount of salmon sperm DNA used as a blocking agent.
• Blocking is accomplished inside the hybridization chamber for duration of
2-3 hours.
Hybridization with DNA Probe
The radioactive DNA probes are implied for the process of hybridization of DNA
in the membrane. The membrane is exposed to a layer of paraffin oil at a
temperature of 45°C. The probe solution is applied after incubating the membrane
in SSC buffer and then allowed for hybridization. Followed by incubation in probe
solution the hybridization step is terminated after 4 hours. This step can be extended
to an overnight duration.
Northern Blot Method
This method involves the analysis of gene expression detected by probe
hybridization with the isolated mRNA transcript obtained from the analyte tissue.
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This method was applied by a group of scientist namely James Alwine, David Screening of
Recombinants, Blotting
Kemp, and George Stark in the year 1977. The name was derived from its Techniques and Blotting
Techniques
resemblance with the southern blot method of DNA hybridization. The method
involves extraction of RNA from the tissue followed by separation of mRNA by
NOTES
using oligo-dT cellulose chromatography. This process of separation preferentially
binds to the mRNA containing Poly-A tail. Followed by RNA extraction and
isolation of mRNA, they are separated by agarose gel electrophoresis. The process
of blot transfer into a nitrocellulose membrane and probe hybridization step remains
similar to that of southern blot method. The nylon membrane is positively charged
and binds to negatively charged RNA molecules. The transfer buffer preferably
contains formamide which can lower the annealing temperature of the probe-RNA
binding (Refer Figure 10.4).
Procedure for Northern Blot
• The tissue is used for RNA extraction by using standard RNA isolation kit
available commercially.
• The mRNA content is isolated from the heterogeneous mixture of RNA
obtained from the tissue. The purification method commonly implied involves
oligo-dT cellulose chromatography. In this method the Poly-A tail containing
mRNA adheres to the cellulosic phase of oligo-dT chain.
• The isolated RNA is separated through standard procedure of agarose gel
electrophoresis. The agarose gels separating the RNA molecules contain
formaldehyde or urea as denaturants.
• The RNA molecules separated on the gel are transferred into the blot
membrane by means of capillary transport and ionic interactions.
• The transfer assembly containing the gel, filter paper and nylon membrane
is immersed in the transfer buffer placed within the transfer unit.
• The RNA molecules being transferred to the nylon membrane is fixed with
UV radiation.
• The nylon membrane is incubated with the probe solution which contains
RNA molecules with complementary sequence to that of the gene of interest.
• The labelling of the probe with the transcript of the gene is detected by
autoradiography or chemiluminiscence method.

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Screening of
Recombinants, Blotting
Techniques and Blotting
Techniques

NOTES

Fig. 10.4 Methodology for Northern Blot

10.4 MAPPING OF HUMAN GENES: HUMAN

GENOME PROJECT

Similar to other organisms, the human genome DNA is organized into separate
units of 23 pairs of chromosomes. The 22 pairs of autosomal chromosomes are
accompanied by a set of sex chromosomes, i.e., XX for females and XY for the
males. Each chromosome contains higher order chromatin organization which
involves supercoiled DNA fragment associated with histone molecules. The DNA
fragment consists of 50-250 million nucleotides aligned in each chromosome. The
human genome is thus composed of 3 billion nucleotide pairs present in the
chromosomal DNA. Human genome maps appear in different size, representation
and resolutions. Low resolution physical maps of human genome have been obtained
by the band patterns observed along each of the chromosomes. This simple pattern
can be obtained by physically staining the chromosomes and visualizing in a light
or fluorescent microscope. However, the exact position of the genes cannot be
obtained by the banding patterns and is required to be deciphered by the other
precise methods. The restriction sites present in and around the genes appear to
be polymorphic in the genome of various populations. This polymorphic DNA
sequences are obtained by variations in the restriction enzyme sites and are resolved
by RFLP (Restriction Fragment Length Polymorphism). Several polymorphic DNA
mediated restriction sites have been deciphered by human genome mapping. The
human genome map can be based on the collection of ordered clone of genomic
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fragments which are maintained in a particular library. The highest resolution map Screening of
Recombinants, Blotting
of the human genome is the nucleotide sequences where the exact position of the Techniques and Blotting
genes is deciphered. Techniques

Human Genome Project and Physical Mapping of Human Genes NOTES

The size and complexity of the human genome imposes various challenges in
deciphering its complete sequence. The International Human Genome Sequencing
Consortium (IHGSC) has attempted to construct the map of the whole human
genome, thus facilitating the selection of clones. This attempt shall successfully
enable to assemble the sequenced regions of the genome. The human genome
project was an international initiative of 15 years duration which was declared to
be complete on April, 2003. The main objective of the project was to decipher
the nucleotide sequence of the human genome Euchromatin regions. The
Euchromatin part comprises of 92% of the entire genome.
• The construction of the whole genome Bacterial Artificial Chromosome
(BAC) has been enabled to facilitate the cloning of the whole genome.
• The method of BAC cloning has been assisted with the integration of
sequence data with the map.
• The IHGSC has enabled the method of hierarchical mapping and sequencing
strategy to complete the genome project.
• The sequencing method implies a clone based approach which involves
generation of overlapping series of clones for the entire human genome.
• The clones are fingerprinted on the basis of the restriction digestion fragments.
• Followed by the above step the clones are selected for shotgun sequencing.
• The whole genome sequence is assembled by overlapping the clone
sequences.
• The clone based map has enabled the recognition of sequences for a larger
segment of the genome which possesses repetitive regions.
• The entire size of the human genome appears to be 3.2 Giga bases. This
results in the analysis of a larger number of clones at a time.
• Maps based on the sequenced tagged sites provide better coverage of the
genome sequencing.
• The construction of P1-Artificial Chromosome (PAC) and BAC have enabled
successful completion of the sequencing project.
• The genome wide data of BAC libraries were used to obtain fingerprint
data of the genome fragments.
• The contigs were merged together to obtain the overlapping sequences
from the fragmented clone sequences
• The BAC fingerprinting initiative was completed between the years 1998-
99 when around 300,000 BACs were fingerprinted from the RPCI-11
library.
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Screening of • A 121-lane agarose gel format was prepared to obtain the fingerprint pattern
Recombinants, Blotting
Techniques and Blotting of the clones obtained from the BAC.
Techniques
• 97.5% of the clones of the total BAC clone genome sequence were obtained
from the RPCI-11 library.
NOTES
• The contigs were aligned according to the expected size of the chromosome

Check Your Progress

4. What is western blot method?
5. Briefly explain concentrating of protein aliquots.
6. Give the methodology for western blot.
7. How is DNA seperated by agarose gel electrophoresis?
8. Define human genome.

10.5 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. The process of screening enables to find the gene of interest present in the
cloning vector of a particular colony. The recombinant vectors containing
the DNA fragment along with marker antibiotic gene are suitable to be
grown in antibiotic supplemented medium. Bacterial and phage colonies
appear to exhibit definite plaqued appearance which is further screened by
probe hybridization or immunological screening. The mechanisms of some
of the methods of screening of recombinants are mainly based on changes
in phenotypic expression of characters.
2. In the direct selection method the clone obtained contain the required gene
inserts. This conventional method is faster does not usually obtain false
positive results. In the process of selection of clone from a colony a random
physical method can also be used to cleave DNA into fragments. The DNA
in question can be either genomic DNA or a clone obtained from Yeast
Artificial Chromosome (YAC).
3. The insertion inactivation method can be successfully implied on plasmids
containing antibiotic resistance genes. The pBR322 plasmid contains two
antibiotic resistance genes for Ampicillin and Tetracycline. The specific region
of the plasmid known as ScaI accommodates the DNA fragment of the
foreign gene. Insertion of the gene in ScaI region results inAmpicillin sensitivity
and tetracycline resistance in the recombinant plasmid. The screening of
recombinant plasmids are accomplished by plating the recombinant
Escherichia coli cells in Tetracycline supplemented medium.
4. Western blot method is applied for immunological detection of protein
molecules priorly separated by PolyAcrylamide Gel Electrophoresis
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 (PAGE). This method enables to detect a particular protein from a mixture Screening of
Recombinants, Blotting
of proteins. The major steps of this method involve: Techniques and Blotting
Techniques
• Separation of protein molecules on the basis of mass of the polypeptides
• Transfer of the proteins into the nitrocellulose or PVDF membrane (blot NOTES
transfer)
• Immunological detection of the proteins using primary and secondary
antibodies.
5. Protein aliquots obtained may have variable protein content (mg/ml)
depending upon the age and type of plant tissues used. However gel
electrophoresis requires good amount of protein content (25-100 µg)
required to be loaded in electrophoresis gels having limitations in the volume
of wells which vary from (30-50 ml). Thus, to reduce water content and
volume of protein aliquots, supernatants obtained are concentrated using
Vivaspin 2 kDa MWCO columns (GE Health Care, UK) at 10,000 g.
Prior to concentrating, the columns are saturated with homogenisation buffer
for 15 minutes at 10,000g to avoid drying of membrane.
6. Sample Preparation for Protein Homogenate: The process of protein sample
preparation includes selection of correct lysis buffer functioning in a proper
range of pH. Separation of membrane proteins requires the application of
strong denaturants like dithioerythritol, sodium dodecyl sulphate or CHAPS
buffer. The extraction of cytosolic proteins can be accomplished by using
sodium-phosphate or Tris-HCl buffer. The lysis buffers should preferably
contain protease inhibitors which shall prevent the process of protein
degradation. beta-mercaptoethanol, or DTT is used in the lysis buffer which
cleave the sulphide bonds present between the cysteine residues of the
protein subunits. Glycerol is commonly used in the laemmli buffer which
acts as an osmoticum and allows the samples to sink in the wells of the
stacking gel.
7. Agarose gel pore size is suitable for separation of DNA varing in range (0.7
kb to 25 kb). Agarose is a linear polymer of D and L galactose residues
alternately joined by glycosidic linkage. The pores in between the agarose
gel matrix helps in efficiently sieving the DNA molecules. The molecular size
of DNA and the percentage of agarose (pore size) are the prime factors
regulating migration rate of DNA in agarose gel electrophoresis. However,
the form of DNA (super helical, nicked circular or linear) also affects the
rate of migration. The voltage applied along with the ionic strength of
electrophoresis buffer affects DNA mobility.
8. The human genome DNA is organized into separate units of 23 pairs of
chromosomes. The 22 pairs of autosomal chromosomes are accompanied
by a set of sex chromosomes, i.e., XX for females and XY for the males.
Each chromosome contains higher order chromatin organization which
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Screening of involves supercoiled DNA fragment associated with histone molecules. The
Recombinants, Blotting
Techniques and Blotting DNA fragment consists of 50-250 million nucleotides aligned in each
Techniques chromosome. The human genome is thus composed of 3 billion nucleotide
pairs present in the chromosomal DNA. Human genome maps appear in
NOTES different size, representation and resolutions.

10.6 SUMMARY

• The process of screening enables to find the gene of interest present in the
cloning vector of a particular colony.
• The recombinant vectors containing the DNA fragment along with marker
antibiotic gene are suitable to be grown in antibiotic supplemented medium.
• Bacterial and phage colonies appear to exhibit definite plaqued appearance
which is further screened by probe hybridization or immunological screening.
• The mechanisms of some of the methods of screening of recombinants are
mainly based on changes in phenotypic expression of characters.
• In the direct selection method the clone obtained contain the required gene
inserts. This conventional method is faster does not usually obtain false positive
results.
• In the process of selection of clone from a colony a random physical method
can also be used to cleave DNA into fragments. The DNA in question can
be either genomic DNA or a clone obtained from Yeast Artificial
Chromosome (YAC).
• The larger fragments of DNA can be inserted into cloning vectors with
lower accommodating capacity. DNA sequencing can be accomplished by
shot gun cloning method where the sequencing is completed in two or three
halves of 600-700 bases in each part.
• The insertion inactivation method can be successfully implied on plasmids
containing antibiotic resistance genes. The pBR322 plasmid contains two
antibiotic resistance genes for Ampicillin and Tetracycline.
• The specific region of the plasmid known as ScaI accommodates the DNA
fragment of the foreign gene. Insertion of the gene in ScaI region results in
Ampicillin sensitivity and tetracycline resistance in the recombinant plasmid.
The screening of recombinant plasmids are accomplished by plating the
recombinant Escherichia coli cells in Tetracycline supplemented medium.
• Separation of membrane proteins requires the application of strong
denaturants like dithioerythritol, sodium dodecyl sulphate or CHAPS buffer.
The extraction of cytosolic proteins can be accomplished by using Sodium
Phosphate or Tris-HCl buffer.
• The Lysis buffers should preferably contain protease inhibitors which shall
prevent the process of protein degradation.
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• The beta-Mercaptoethanol, or DTT is used in the Lysis buffer which cleave Screening of
Recombinants, Blotting
the sulphide bonds present between the cysteine residues of the protein Techniques and Blotting
subunits. Techniques

• Glycerol is commonly used in the Laemmli buffer which acts as an Osmoticum NOTES
and allows the samples to sink in the wells of the stacking gel.
• Samples are homogenized in Tris-urea buffer (50 mM Tris, pH 7.5, containing
9 M urea and filtered through 4 layers of muslin cloth.
• The filtrate thus obtained is centrifuged at 10,000 g at 4 °C for 20 minutes.
The oil body pads collected is subsequently resuspended in 1.25 mL
NaHCO3 (0.1 M) per gram of tissue and incubated at 4°C for 30 minutes
and again centrifuged at 10,000 g for 20 minutes at 4°C.
• The oil body pads are then resuspended in excess Tris-sucrose buffer (20
mM Tris, 0.2 M Sucrose, pH-7.5) to wash out excess bicarbonate.
• After centrifugation at 10,000 g for 20 minutes at 4°C, the oil body pads
are resuspended in the same buffer. In order to solubilize oil body membrane
proteins, oil body suspension washed in Tris-sucrose buffer is extracted
four times with five volumes of diethyl ether.
• Residual diethyl ether is evaporated from each sample at room temperature
while vortexing continuously.
• The residues containing oil body membrane proteins is suspended in Tris-
sucrose buffer. 160 µL of this suspension is mixed with 40 mL of 10% SDS
solution.
• The samples are heated at 90 °C in a water bath for 30 minutes in order to
solubilise membrane proteins, and centrifuged for 15 minutes at 7000 g and
4°C. Oil body membrane proteins in the supernatant are quantified according
to Markwell’s method of protein quantification.
• Protein aliquots obtained may have variable protein content (µg/mL)
depending upon the age and type of plant tissues used.
• Gel electrophoresis requires good amount of protein content (25-100 µg)
required to be loaded in electrophoresis gels having limitations in the volume
of wells which vary from (30-50 ml).
• To reduce water content and volume of protein aliquots, supernatants
obtained are concentrated using Vivaspin 2 kDa MWCO columns (GE
Health Care, UK) at 10,000 g. Prior to concentrating, the columns are
saturated with homogenisation buffer for 15 minutes at 10,000 g to avoid
drying of membrane.
• Protein homogenates after concentrating require purification steps in order
to reduce phenolic and lipidic constituents. These impurities if present often
interfere with various stains used in protein staining gels.
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Screening of • Phenolic compounds along with degraded DNA or RNA products often
Recombinants, Blotting
Techniques and Blotting cause streaking and intense background stains in the electrophoretograms.
Techniques
• Concentrated homogenates obtained are proceeded for delipidation steps
by acetone washings. Homogenates are incubated with 100% acetone (1:3
NOTES
v/v) overnight at -20°C followed by gentle vortex and centrifugation at
10,000 g at 4°C for 20 minutes.
• Supernatants containing all organic contaminants mixed with acetone are
discarded and the protein pellets obtained are suspended in 90% acetone
followed by incubation at -20°C for 20 minutes.
• After incubation pellets are collected by centrifugation (10,000g for 20
minutes at 4°C) followed by two more acetone (90%) washings. Pellets
obtained after acetone washings are then air dried to remove all traces of
acetone vapours and suspended in 200 µL homogenisation buffer. Protein
suspensions are then sonicated for 5 minutes and centrifuged at 10,000 g
for 20 minutes at 4°C.
• Alternatively TCA and chloroform/methanol mixture can also be used to
precipitate proteins and dissolve Lipidic constituents.
• Bradford’s method of protein quantification is one of the most widely used
simple protocols for calculating protein concentrations in terms of mg/mL in
sample aliquots. Denaturants like urea used in protein solubilisation buffer
(applicable for extraction of membrane proteins) often interfere with binding
to the dye and liberating false results.
• Standard curve of Bradford’s estimation involves Bovine Serum Albumin
(BSA) solution dissolved in 0.75 M NaCl solution at a concentration of
1mg/mL. Further dilutions (100, 80, 60, 40 and 20 µg/mL) are prepared
with NaCl solutions.
• Standard and sample aliquots are incubated separately with Bradford’s
reagent in dark at 25ºC for 20 minutes followed by which absorbance is
recorded at 595 nm.
• Blank containing only Bradford’s reagent and homogenisation buffer is used
as a control to set reference or zero in spectrophotometer.
• The standard curve plotted with absorbance and concentration parameters
is used to derive the value of 0.1 absorbance value equivalent to the amount
of protein in mg. The absorbance thus obtained from samples is then
calculated for amount of protein present.
• The DNA are transferred onto a membrane and immobilized before
hybridization with the fluorescent probe.
• The process of elution of DNA from the gel and designing of the probe are
important components of the method. In comparison with other techniques
southern blot hybridization offers better resolution in terms of DNA detection.
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 • The main components of the method involve extraction of DNA, digestion Screening of
Recombinants, Blotting
of DNA by restriction enzymes, separation by agarose gel electrophoresis Techniques and Blotting
Techniques
followed by transfer of DNA into the nitrocellulose membrane.
• White colonies represent transformants with GOI incorporated in the vector. NOTES
• The insertion inactivation method can be successfully implied on plasmids
containing antibiotic resistance genes.
• Plating in two different media will result in comparison of the colonies unable
to grow in ampicillin medium.
• The cI gene inactivation method implies the principle of disruption of cI
factor present in the bacteriophage.
• Functional complementation is the process of compensating the loss of
function of a mutant host cell.
• The vector which contains the gene insert shall complement the mutant
auxotroph and shall be capable of growing in the deficient nutrient medium.

10.7 KEY WORDS

• Functional complementation: Functional complementation is the process

of compensating the loss of function of a mutant host cell.
• Blot: A blot, in molecular biology and genetics, is a method of
transferring proteins, DNA or RNA, onto a carrier.
• Western blot method: The method is applied for immunological detection
of protein molecules earlier separated by PolyAcrylamide Gel
Electrophoresis (PAGE). This method enables to detect a particular protein
from a mixture of proteins.

10.8 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. Write in short about screening of recombinants.
2. Give a brief summary of insertion inactivation method.
3. Explain the functioning of blue white screening method.
4. Briefly describe antibiotic resistance and sensitivity method.
5. Distinguish between Western, Southern and Northern blot.
6. What is HGP?
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Screening of Long Answer Questions
Recombinants, Blotting
Techniques and Blotting
Techniques 1. Explain the methods of recombinant clone screening.
2. What do you mean by phenotypic expression of character?
NOTES 3. Explain with the help of diagrams the principle and procedure for Western
blotting.
4. Distinguish between southern blot and northern blot.
5. Mention the salient features of Human Genome Project (HGP).

10.9 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 PCR Methods and
BLOCK - IV Primer Designing

MOLECULAR TECHNIQUES
NOTES
UNIT 11 PCR METHODS AND
PRIMER DESIGNING
Structure
11.0 Introduction
11.1 Objectives
11.2 Polymerase Chain Reaction (PCR)
11.2.1 Components of PCR Reaction Procedure
11.2.2 Steps of Polymerase Chain Reaction
11.3 Primer Designing and Optimization
11.3.1 Procedure for Primer Designing
11.4 Variations in PCR
11.4.1 Random Amplification of Polymorphic DNA (RAPD): PCR Variant
11.4.2 Restriction Fragment Length Polymorphisms (RFLP)
11.5 Answers to Check Your Progress Questions
11.6 Summary
11.7 Key Words
11.8 Self Assessment Questions and Exercises
11.9 Further Readings

11.0 INTRODUCTION

The Polymerase Chain Reaction (PCR) involves selective amplification of a part

of the DNA or RNA segment, specifically a gene fragment. PCR is a widely applied
method in molecular biology which enables gene cloning by preparing multiple
amplicons or copies of a particular gene. In molecular biology, an amplicon is a
piece of DNA or RNA that is the source and/or product of amplification or
replication events. It can be formed artificially, using various methods including
Polymerase Chain Reactions (PCR) or Ligase Chain Reactions (LCR), or naturally
through gene duplication. In 1976, the heat stable form of DNA polymerase was
isolated from thermophile Thermus aquaticus, a species of bacteria that can tolerate
high temperatures. It is one of several thermophilic bacteria that belong to the
Deinococcus–Thermus group. Over the years of development PCR has
revolutionized with variations to clone gene segments subject to different conditions.
The steps of PCR essentially comprises of 20-40 thermal cycles each of which
comprise of the steps carried out at specific temperatures and are namely
denaturation, annealing and extension. The PCR process was originally developed
to amplify short segments of a longer DNA molecule. A typical amplification reaction
includes target DNA, a thermostable DNA polymerase, two oligonucleotide
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PCR Methods and primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium.
Primer Designing
Once assembled, the reaction is placed in a thermal cycler, an instrument that
subjects the reaction to a series of different temperatures for set amounts of time.
The possibilities of intermolecular or intramolecular attraction among the
NOTES
primer sequences may result in the formation of hairpins, dimers or repeats and
runs. The RAPD analysis is a PCR-based method which involves amplification
of a target DNA in exponential forms. The primer used is arbitrary in nature and
involves usual PCR method of denaturation, annealing and elongation in the
presence of DNA polymerase and dNTPs. RAPD markers are of short length
(10 nucleotides) and require a single primer. The process of RAPD does not
involve any requirement of blotting or hybridization steps. The DNA fragments
obtained from RAPD can be further cloned and sequenced to produce other
types of markers like SNP. RFLP is a PCR based method which exhibits
differences in homologous DNA that can detect differences in the sequence solely
on the basis of variations in the restriction endonuclease sites. Mostly RFLP are
co-dominant markers and locus specific. The differences in the genotypes can
be analysed by RFLP.
In this unit, you will study about the Polymerase Chain Reaction (PCR),
gene amplification, primer designing, optimization, variation in the PCR - RAPD
and RFLP.

11.1 OBJECTIVES

After going through this unit, you will be able to:

• Understand the concept of Polymerase Chain Reaction (PCR)
• Analyse the steps involved in the PCR
• Explain gene amplification
• Discuss the concept of primer designing
• Elaborate on the methodology for variation in the PCR, RAPD and RFLP

11.2 POLYMERASE CHAIN REACTION (PCR)

The Polymerase Chain Reaction (PCR) involves selective amplification of a part

of the DNA or RNA segment, specifically a gene fragment. PCR is a widely
applied method in molecular biology which enables gene cloning by preparing
multiple amplicons or copies of a particular gene. In molecular biology, an amplicon
is a piece of DNA or RNA that is the source and/or product of amplification or
replication events. It can be formed artificially, using various methods including
Polymerase Chain Reactions (PCR) or Ligase Chain Reactions (LCR), or naturally
through gene duplication. PCR has important applications in gene cloning,
sequencing and target detection. The process of DNA replication is catalyzed by
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the activity of Taq polymerase, primers and dNTPs. The reaction carried out by PCR Methods and
Primer Designing
the method of PCR is accomplished by thermal cycles (heating and repeated cooling
to allow primer hybridization and replication).
In the conventional method of DNA replication in vitro, primers and templates
were used to obtain replicated copies of DNA. However, the DNA polymerase NOTES
used in the process was unable to withstand the high temperature necessary for
DNA unwinding. Later in 1976, the heat stable form of DNA polymerase was
isolated from thermophile Thermus aquaticus which can be stable at temperatures
up to 95°C. Thermus aquaticus, is a species of bacteria that can tolerate high
temperatures. It is one of several thermophilic bacteria that belong to the
Deinococcus–Thermus group. Using this enzyme Kary Mullis reported the
application of polymerase chain reaction technique in the year 1985. Over the
years of development PCR has revolutionized with variations to clone gene segments
subject to different conditions.
The steps of PCR essentially comprises of 20-40 thermal cycles each of
which comprise of the steps carried out at specific temperatures and are namely
denaturation, annealing and extension. The PCR process was originally developed
to amplify short segments of a longer DNA molecule. A typical amplification reaction
includes target DNA, a thermostable DNA polymerase, two oligonucleotide
primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium.
Once assembled, the reaction is placed in a thermal cycler, an instrument that
subjects the reaction to a series of different temperatures for set amounts of time.
Figure 11.1 illustrates the gradual breakthrough from the discovery of DNA
structure to the invention of modern PCR, i.e., the advancements in the PCR
techniques.
Year The Gradual Breakthrough from the Discovery of DNA Structure to the Invention
of Modern PCR
1950 Discovery of Mechanism of DNA Replication by Arthur Komberg. He discovered the
First DNA Polymerases and other factors, like Helicase and Primers.
1796 Isolation of thermostable DNA Polymerase from Thermus aquaticus.
1983 Kary Banks Mullis synthesized DNA Oligo probes for Sickle Cell Anaemia mutation.
1983 Repeated thermal cycling was first used for small segment of Cloned Gene.
1984 Kary Banks Mullis and Tom White tried designed experiments to test PCR on Genomic
DNA but the amplified product was not visible in Agarose Gel.
1985 Patent was filed for PCR and its applications focusing on Sickle Cell Anaemia mutation.
1985 The use of thermostable DNA Polymerase in PCR was started. Only two enzymes, Taq
and Bst polymerases were known at that time. Out of the two, the Taq polymerase was
found more suitable for the PCR methodology.
1985 First announcement of PCR technique was done in the Salt Lake City (US).
1985-1987 Development of precise techniques and instruments for PCR and its Reagents.

Fig. 11.1 Advancements in the PCR Techniques

11.2.1 Components of PCR Reaction Procedure

The major components and reagents required to perform a PCR is as follows:
1. Target DNA: The template DNA involves the segment of the gene required
to be amplified.
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PCR Methods and 2. Primers: The primers are short nucleotide sequences complementary to
Primer Designing
the gene sequence at the 5´ end. There are two sets of primers called
forward primer and reverse primer complementary to the sense and
antisense strand of the DNA, respectively.
NOTES
3. DNA Polymerase: Taq polymerase is usually preferred for PCR methods
which is attributed to its heat stable nature.
4. dNTPs: The deoxynucleotide triphosphate (dNTP) molecules are required
in an optimum concentration such that the reaction proceeds in a forward
equilibrium. The dNTPs function as building blocks for the synthesizing
strand of DNA where they join by phosphor-diester linkage and form
hydrogen bond with the complementary base in the mother strand.
5. Reaction Buffer: It maintains the correct pH necessary for the reaction
conditions.
6. Cofactor: Taq polymerase requires the presence of Mg2+ as a cofactor to
maintain its catalytic activity.
Table 11.1 illustrates the components of a PCR, i.e., reagents and amount required.
Table 11.1 Components of a PCR

11.2.2 Steps of Polymerase Chain Reaction

The steps of PCR essentially comprises of 20-40 thermal cycles each of which
comprise of the steps carried out at specific temperatures and are namely
denaturation, annealing and extension. The initial step of denaturation occurs at
a higher temperature >90°C. Then further cooling is done at a temperature of
4°C to store the amplicons. The temperature of each stage and the duration of
reactions depend upon various factors, such as the type of DNA polymerase
used, nature of DNA (GC content), melting temperature of primers, concentration
of dNTPs and cofactors in the reaction mixture. Figure 11.2 illustrates the
temperature profile for various steps of PCR while the Figure 11.3 shows the
various steps of PCR.

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 PCR Methods and
Primer Designing

NOTES

Fig. 11.2 Temperature Profile for Various Steps of PCR

Fig. 11.3 Steps of PCR Self-Instructional

Material 229
PCR Methods and Denaturation: The initial process of denaturation involves heating of the
Primer Designing
reaction mixture at 95°C for a duration of 10 minutes. Specially engineered DNA
polymerase called Hot Start Taq Polymerase may require a higher denaturation
temperature. The process of initial denaturation helps in unwinding of the genomic
NOTES DNA and preferably exposes the gene segment accessible to the primer. Further
denaturation is accomplished at a temperature of 94–96°C for 20-30 seconds.
This results in the unwinding of the required template DNA caused by the disruption
of hydrogen bonds between the complementary bases which yield single stranded
DNA molecules. Figure 11.4 illustrates the denaturation of template DNA.

Fig. 11.4 Denaturation of Template DNA

Annealing: Followed by the process of denaturation, the annealing step is important

for the binding of the primer to the single stranded template DNA. The binding at
specific region of the DNA is important for obtaining correct amplicons. This,
however, depends mainly on the choice of primer with required complementary
sequence. The temperature is lowered to 50–65°C for a duration of 20–50 seconds
which initiates the process of annealing. The temperature used for annealing is
usually kept a few degrees lower than the Tm (melting temperature) of the primers.
A high degree of sequence similarity between the primer and DNA template
effectively causes the process of annealing to be successful. The DNA polymerase
activity initiates replication form the 3´ end and proceeds towards the 5´ end.
Figure 11.5 illustrates the annealing process of DNA with a primer.

Fig. 11.5 Annealing of DNA with a Primer

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Extension: This step involves elongation of the new DNA fragment synthesized PCR Methods and
Primer Designing
as a complementary strand to that of the mother strand. The DNA polymerase
catalyzes the addition of dNTPs to the 3´ end of the primers. The nature of DNA
polymerase used determines the temperature required for the elongation process.
Taq polymerase catalyzes the addition the dNTPs with their 5´ phosphate end NOTES
joined by phosphor-diester bond to the 3´ hydroxyl end of the extending DNA
strand. The DNA polymerase can effectively add 1000 nucleotides in a time span
of 1.5 minutes. In the presence of optimum conditions during elongation step the
DNA fragment increases its copies in exponential number. One of the major
limitations of Taq polymerase is that it lacks the 3´-5´ exonuclease activity which
lowers the fidelity of replication. The error in the incorporation of nucleotide during
replication may account to 1 in 9000 nucleotides. Figure 11.6 represents the
elongation of New DNA Strand.

Fig. 11.6 Elongation of New DNA Strand

Final Elongation: This step is essentially performed for 1 minute at a temperature

of 75°C. This step is accomplished at the end of the PCR to ensure the replication
of any left out single stranded DNA molecules. The final hold stage at the end of
final elongation occurs by lowering the temperature to 4°C which helps in the storage
of the reaction mixture. After the accomplishment of around 30 cycles the amplicons
are expected to contain several copies of the desired gene sequence. To confirm
the presence of amplicons resulting from replication of the desired gene, the PCR
products are resolved on an agarose gel electrophoresis which results in the
appearance of DNA band comparable with marker DNA ladders. The gene
amplicon should be represented by the desired size of band in kilo base pairs
(kbp). However, exact confirmation of the amplicons can only be obtained by the
process of gene sequencing.

11.3 PRIMER DESIGNING AND OPTIMIZATION

The success of obtaining correct amplicons of gene mainly depends upon the choice
of correct primers. The primers to function effectively, certain properties are required
to be taken into consideration as follows.
1. The primers should essentially be of 17-30 nucleotides in length. The RAPD
primers should be around 10-12 nucleotide long.
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PCR Methods and 2. The nucleotide composition of the primer should comprise around 50%
Primer Designing
GC content or 40-60%. The GC triple hydrogen bonds are stronger than
AT pairs thus increasing the melting point of the primer. Primers with low
GC content should be effectively longer to avoid low melting temperature.
NOTES
3. The calculation of the melting point of the primer is regulated by the percentage
of AT and GC present in the primer. The formula is as follows,
Tm = 4°C x (Number of G’s and C’s in the Primer) + 2°C x (Number
of A’s and T’s in the Primer)
4. Both forward primer and reverse primer should have the similar Tm value.
Optimization of the primer to be selected for replication among a set of
primers should possesses the highest Tm.
5. Primer sequences with more than 3 nucleotides repetitive in the sequence
should be avoided. This results in the formation of secondary structure (hairpin
loop) which are undesirable for the PCR method.
6. The forward primer and reverse primer should not contain any base
complementarities to each other.
7. Primers with Tm­ in the range of 52-58°C provides better results in PCR. A
maximum allowable difference of Tm between the forward primer and reverse
primer can be of 2°C. Higher differences in this case may result in annealing
inefficiencies.
8. The number of GC bonds present at the 3´ end of the primer should not be
repetitively more than three. This would otherwise form non-specific
secondary structures or GC clamps.
9. The possibilities of intermolecular or intramolecular attraction among the
primer sequences may result in the formation of hairpins, dimers or repeats
and runs. The later results from long repeats of nucleotides in the primer
sequence which is not desired to exceed more than four in consecutive
numbers.
10. Primers should be designed in a manner so as to avoid minimum sequence
homology with the regions of the DNA template other than the gene sequence.
This shall otherwise cause non-specific binding.

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Following Figure 11.7 specifies the relation of primer length and GC content. PCR Methods and
Primer Designing

NOTES

Fig. 11.7 Relation of Primer and GC Content

11.3.1 Procedure for Primer Designing

The process of primer designing can be accomplished by referring to the sequence

form literature or from databases or software. The NCBI primer blast is one of
the convenient sources to design the primers. However, apart from NCBI, Primer
3Plus can also be used for obtaining desirable sequences of primers. The nature of
primer sequence also depends upon the method of cloning preferred in the PCR
method. In the process of restriction free cloning the primer sequence is expected
to possess both vector and gene specific sequences which also contain restriction
sites present in it. In restriction digestion based cloning the primer contains only
the gene specific sequence along with restriction sites. Apart from Primer 3 online
tool, Beacon 5 software is also effective in delivering the sequences of forward
primers and reverse primers. The online applications for primer designing usually
require the DNA sequences necessary to be referred to design the primers. The
important parameters or settings considered for designing the primers are as follows.
1. Primer Size: Minimum 14-18 nu; Optimum-20 nu; Maximum-27 nu.
2. Primer Tm: Minimum 57°C; Optimum-60°C; Maximum-63°C
3. Primer GC%: Minimum 30%; Optimum-50%; Maximum-60%
4. Concentration: The concentration of monovalent and divalent cations
should be - µM.
5. Concentration of dNTPs: As Per Requirement.
6. Advanced Settings: Various other parameters like maximum repeat
mispriming, maximum allowable 3´-self complementarity, leader sequence
(bp), spacing and interval sequence (bp).
7. Exon-Exon Gap: Inclusion or intron inclusion may also be required for the
mRNA sequence being used as a PCR template. The length of intron may
be required in this case. Primer specificity stringency is also an important
parameter which explains the number of mismatches to unintended targets
or at the last base pairs in the 3´ end.
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PCR Methods and Figure 11.8 specifies the annealing process of the primer with template DNA.
Primer Designing

NOTES

Fig. 11.8 Annealing of Primer with Template DNA

Figure 11.9 illustrates the self-dimer and hairpin, the possible secondary structures
formed due to self-complementarity of primers.
Self-Dimer

Hairpin

Fig. 11.9 Possible Secondary Structures Formed due to

Self-Complementarity of Primers
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 PCR Methods and
The following screenshots of Primer-BLAST software and Primer3Plus software Primer Designing
will help you understand that how to use these software for primer designing and
accomplish the general settings. These software helps in finding specific primers.
Primer-BLAST software helps in finding specific primers. NOTES

Primer3Plus software – general settings for primer designing that helps in picking
specific primers from a DNA sequence.

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PCR Methods and Primer3Plus software – advanced settings for primer designing that helps in picking
Primer Designing
specific primers from a DNA sequence.

NOTES

11.4 VARIATIONS IN PCR

The versatility of Polymerase Chain Reaction (PCR) has led to a large number of
variants of PCR.
Reverse Transcription PCR (RT-PCR)

In this type of PCR initially a RNA template is reverse transcribed into the
complementary DNA catalyzed by the activity of reverse transcriptase. The
produced cDNA is amplified by the help of PCR. Various sources like Avian
Myeloblastosis Virus (AMV), Moloney Murine Leukemia Virus
(MMLV or MuLV) are implied to obtain the enzyme reverse transcriptase. The
primers used for RT–PCR may be random primers and oligo-dT primer or primers
specific to the sequences, sequence specific primer. Certain forms of DNA
polymerase possesses reverse transcriptase activity and require the presence of
Mn2+ as a cofactor. RT-PCR finds application in monitoring gene expression
associated with genetic disorders, stress induction and in monitoring developmental
stages. Figure 11.10 illustrates the steps for the reverse transcriptase PCR.

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 PCR Methods and
Primer Designing

NOTES

Fig. 11.10 Reverse Transcriptase PCR Steps

Real Time PCR (Quantitative PCR)

This quantitative method involves measuring the relative amounts of target sequence
in a sample. The amount of DNA generated can be quantified by the assistance of
a fluorescent probe. In this type of PCR, DNA amplification is measured at the
end of each cycle. The increasing fluorescence intensity indicates the exponential
increase in amplicons. The increase of fluorescent intensity beyond the threshold
level in comparison to the basal level indicates the threshold cycle. A standard
curve of CT­ is allowed to be detected by the fluorescent probe which correlates
with the amount of DNA or cDNA measured within a tissue. The fluorescent dye
used is commonly SYBR Green I which intercalates between the base pairs of the
DNA strand. The SYBR Green I is an asymmetrical cyanine dye used as a nucleic
acid stain in molecular biology. After binding to the DNA it increases the fluorescence
intensity several folds in comparison to the unbound state. Alternately
TaqMan probes or Molecular BeaconsTM can also be implied which contain primer
or short oligonucleotide sequences complementary to the target DNA. The probes
exhibit negligible fluorescence in unbound conditions. Thus, the fluorescence
intensity obtained is directly proportional to amount of DNA produced in the cycles
of amplification. Scorpion primers are similar to the Molecular Beacons™, however,
they possesses a PCR primer sequence. During PCR amplification the beacon
fragment binds to the nascent DNA thus separating the fluorophore from the
quencher. A PCR-blocker is used to prevent the binding of the primer to hairpin
structure. The kinetics of scorpion primer binding with the PCR product is stable
than that of other probes and exhibit prominent fluorescent signal. Figure 11.11
illustrtaes the real time PCR.

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PCR Methods and
Primer Designing

NOTES

Fig. 11.11 Real Time PCR

Hot Start PCR

The fact that lowering temperature in PCR may lead to decrease in the activity of
Taq polymerase. In certain conditions primer binding at low temperatures is often
non-specific in nature. Hot Start PCR involves mitigation of primer annealing or
dimer formation. The method involves withholding the reaction components like
DNA polymerase, PCR primers, dNTPs and cofactors from being involved in
reaction until the temperature of the first cycle reached to as high as 95°C, i.e.,
higher than the annealing temperature. The process can also be performed manually
by attaining the temperature to the range prior to addition of DNA polymerase.
Various reagents or wax compounds may separate the enzyme from being activated
at lower temperatures. Genetically engineered DNA polymerase may be used in
Hot Start PCR which exhibits absence of activity at lower temperature.
Colony PCR
In this method the bacterial colonies are directly used for PCR amplification. This
facilitates in screening of recombinant colonies with the transformed DNA vector
constructs. Colony samples are collected with a sterile pipette tip or toothpick
and then transferred to a PCR mix. The reaction in the PCR machine is run at 95-
100°C for an extended duration which results in cell disruption. Usually the
temperature is only raised to 100°C with genetically engineered DNA polymerase
being used in the PCR mix. This method is widely used in molecular biology and it
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verifies the presence of the gene insert in the vector, its orientation and size. This PCR Methods and
Primer Designing
method is less tedious in terms of sample preparation. There is no requirement of
plasmid or genomic DNA isolation, southern blotting or restriction digestion to be
performed. Large colonies can be effectively screened for the presence of
recombinant cells. NOTES

Nested PCR
This modification of PCR aims to prevent the presence of unwanted amplicons
produced from non-specific binding of primers to the DNA template. This method
implies two sets of primers called inner primers and outer primers, respectively.
The DNA target sequence of the outer primer is present in the DNA template.
The outer primer is implied first in the reaction. Followed by a single cycle of
amplification the inner primer is added. The amplicons produced in the first cycle
function as targets for the inner primer. The sensitivity of this method increases by
several folds. The correct amplification in the first cycle will selectively amplify it in
the subsequent cycle. Figure 11.12 illustrates the nested PCR.

Fig. 11.12 Nested PCR

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PCR Methods and Inverse PCR
Primer Designing
The conventional PCR method bears limitations like the 5' and 3' ends should
possesses known sequences. In inverse this limitation is overcome where
NOTES amplification is possible with a single known internal sequence. The primers are
oriented in the reverse direction in respect to the normal direction. The template in
this type of reaction is a self-ligating restriction fragment which forms a circle. This
kind of PCR involves restriction endonuclease mediated digestion of DNA fragment
followed by induction of self-ligation process to form a circular DNA. Followed
by circularization it is further digested with a known restriction endonuclease to
generate a nick in the known internal sequence. The resultant cut fragments have
their terminal séances known and are used as templates for amplification. Figure
11.13 illustrates the process of inverse PCR.

Fig. 11.13 Schematic Representation of Inverse PCR

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11.4.1 Random Amplification of Polymorphic DNA (RAPD): PCR PCR Methods and
Primer Designing
Variant
This method is variation of PCR which develops molecular marker for establishing
genetic maps or to accomplish DNA finger printing. RAPD markers are of short NOTES
length (10 nucleotides) and require a single primer. The method does not require
any prior information on the sequence of DNA obtained from the tissue to be
analyzed. The polymorphism obtained is created due to the relatively variable
DNA sequences between different species, ecotypes or population present as a
result of mutation (chromosomal rearrangements) in between the primer binding
sites. The products of PCR for RAPD analysis are visualized by Agarose Gel
Electrophoresis and after staining with Ethidium Bromide. The process of RAPD
does not involve any requirement of blotting or hybridization steps. Very small
amount of DNA (10 ng/reaction) can be sufficient to undertake this method of
analysis. The primers used are not essentially species specific but universal. The
DNA fragments obtained from RAPD can be further cloned and sequenced to
produce other types of markers like SNP. However, one limitation of RAD is that
it produces dominant markers and does not differentiate allelic variations for a
gene. Thus the markers generated for a set of sample DNA does not differentiate
between homozygous and heterozygous conditions for a particular locus.
Method of RAPD Analysis
1. The RAPD analysis is a PCR-based method which involves amplification
of a target DNA in exponential forms. The primer used is arbitrary in nature
and involves usual PCR method of denaturation, annealing and elongation
in the presence of DNA polymerase and dNTPs.
2. The PCR mix is prepared and applied with requisite temperature
programming.
3. The amplification is performed for the necessary time and aliquots are
obtained.
4. The aliquots containing 3 µl DNA is loaded into the wells of 1.2% agarose
gel in 0.5 X TBE buffer.
5. The gel is run at 554 volts for 4 hours. Followed by electrophoretic separation
the DNA bands are visualized by Ethidium Bromide fluorescence or Silver
staining.
6. The polymorphic band pattern is used a marker for analyzing genomic DNA
or other target DNA among the different samples (population assemblage
or ecotype).
7. The polymorphic DNA forms are eluted and sequenced to obtain information
on the polymorphic markers.
8. The polymorphic regions are further amplified by PCR method to develop
markers.
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PCR Methods and Figure 11.14 illustrates the various steps of RAPD.
Primer Designing

NOTES

Fig. 11.14 Steps of RAPD

Figure 11.15 illustrates the Agarose Gel profile showing Polymorphic DNA Band
Pattern for DNA of different samples.

Fig. 11.15 Agarose Gel Profile Showing Polymorphic DNA Band Pattern for
DNA of Different Samples

11.4.2 Restriction Fragment Length Polymorphisms (RFLP)

This method of PCR-mediated amplification is based on the fragments of genomic
DNA previously digested by restriction enzymes. It produces sets of molecular
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markers necessary for animal or plant genome mapping project. The method is PCR Methods and
Primer Designing
capable of detecting the mutations occurring in each loci which manifests by a gain
or loss in the restriction sites. DNA obtained from homologous chromosomes can
be digested by restriction enzyme which shall yield polymorphism in the DNA
fragments obtained by electrophoresis. RFLPs are sensitive and accurate PCR- NOTES
based methods which can possibly detect single nucleotide mutation, deletion or
insertion within 1-100 bp of DNA. This method, however, requires higher amount
of DNA, and specific probe libraries. The DNA patterns obtained may exhibit
low polymorphism. Figure 11.16 illustrates the RFLP profile used as co-dominant
markers for a Locus.

Fig. 11.16 RFLP Profile used as Co-Dominant Markers for a Locus

Method for RFLP

1. RFLP is a PCR based method which exhibits differences in homologous
DNA that can detect differences in the sequence solely on the basis of
variations in the restriction endonuclease sites.
2. Mostly RFLP are co-dominant markers and locus specific.
3. RFLP probes used in experiments are usually labelled DNA sequence that
hybridizes with one or more fragment of digested DNA fragment resolved
by electrophoresis. Usually short cDNA’s are used as RFLP probes.
4. The differences in the genotypes can be analysed by RFLP.
5. The entire genomic DNA is digested with a methylation sensitive enzyme.
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PCR Methods and 6. The fragmented DNA are amplified using primers in a PCR.
Primer Designing
7. The amplified DNA is separated on an Agarose Gel and observed for
polymorphic patterns of DNA Bands.
NOTES 8. The polymorphic fragments can be eluted, sequenced and cloned in vectors.
9. RFLP probes can be used to perform southern blot hybridization and
confirmation of RFLP markers.
Figure 11.17 illustrates the steps involved for probe based RFLP.

Fig. 11.17 Steps of Probe Based RFLP

Check Your Progress

1. What is Polymerase Chain Reaction (PCR)? What is amplicon?
2. Explain the process of DNA replication using PCR.
3. What are the steps of PCR method?
4. How is correct amplicons of genes obtained?
5. Explain some variants of PCR.
6. What is Random Amplification of Polymorphic DNA (RAPD) method?
7. Define Restriction Fragment Length Polymorphisms (RFLP) method.

11.5 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. The Polymerase Chain Reaction (PCR) involves selective amplification of

a part of the DNA or RNA segment, specifically a gene fragment. PCR is a
widely applied method in molecular biology which enables gene cloning by
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 preparing multiple amplicons or copies of a particular gene. In molecular PCR Methods and
Primer Designing
biology, an amplicon is a piece of DNA or RNA that is the source and/or
product of amplification or replication events. It can be formed artificially,
using various methods including Polymerase Chain Reactions (PCR) or
Ligase Chain Reactions (LCR), or naturally through gene duplication. NOTES
2. PCR has important applications in gene cloning, sequencing and target
detection. The process of DNA replication is catalyzed by the activity of
Taq polymerase, primers and dNTPs. The reaction carried out by the method
of PCR is accomplished by thermal cycles (heating and repeated cooling to
allow primer hybridization and replication).
3. The steps of PCR essentially comprises of 20-40 thermal cycles each of
which comprise of the steps carried out at specific temperatures and are
namely denaturation, annealing and extension. The PCR process was
originally developed to amplify short segments of a longer DNA molecule.
A typical amplification reaction includes target DNA, a thermostable DNA
polymerase, two oligonucleotide primers, deoxynucleotide triphosphates
(dNTPs), reaction buffer and magnesium. Once assembled, the reaction is
placed in a thermal cycler, an instrument that subjects the reaction to a
series of different temperatures for set amounts of time.
4. The success of obtaining correct amplicons of gene mainly depends upon
the choice of correct primers. Hence,
• The primers should essentially be of 17-30 nucleotides in length. The
RAPD primers should be around 10-12 nucleotide long.
• The nucleotide composition of the primer should comprise around 50%
GC content or 40-60%. The GC triple hydrogen bonds are stronger
than AT pairs thus increasing the melting point of the primer. Primers
with low GC content should be effectively longer to avoid low melting
temperature.
• The calculation of the melting point of the primer is regulated by the
percentage of AT and GC present in the primer. The formula is as follows,
Tm = 4°C x (Number of G’s and C’s in the Primer) + 2°C x (Number
of A’s and T’s in the Primer)
• Both forward primer and reverse primer should have the similar Tm value.
Optimization of the primer to be selected for replication among a set of
primers should possesses the highest Tm.
• The forward primer and reverse primer should not contain any base
complementarities to each other.
5. The versatility of Polymerase Chain Reaction (PCR) has led to a large number
of variants of PCR.
• Reverse Transcription PCR (RT-PCR) is type of PCR where initially a
RNA template is reverse transcribed into the complementary DNA
catalyzed by the activity of reverse transcriptase. The produced cDNA
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PCR Methods and is amplified by the help of PCR. Various sources like Avian
Primer Designing
Myeloblastosis Virus (AMV), Moloney Murine Leukemia Virus
(MMLV or MuLV) are implied to obtain the enzyme reverse
transcriptase.
NOTES
• Real Time PCR (Quantitative PCR) method involves measuring the
relative amounts of target sequence in a sample. The amount of DNA
generated can be quantified by the assistance of a fluorescent probe. In
this type of PCR, DNA amplification is measured at the end of each
cycle. The increasing fluorescence intensity indicates the exponential
increase in amplicons.
• Hot Start PCR involves mitigation of primer annealing or dimer formation.
The method involves withholding the reaction components like DNA
polymerase, PCR primers, dNTPs and cofactors from being involved
in reaction until the temperature of the first cycle reached to as high as
95°C, i.e., higher than the annealing temperature.
• Inverse PCR amplification is possible with a single known internal
sequence. The primers are oriented in the reverse direction in respect to
the normal direction. The template in this type of reaction is a self-ligating
restriction fragment which forms a circle.
6. Random Amplification of Polymorphic DNA (RAPD) method is variation
of PCR which develops molecular marker for establishing genetic maps or
to accomplish DNA finger printing. RAPD markers are of short length (10
nucleotides) and require a single primer. The method does not require any
prior information on the sequence of DNA obtained from the tissue to be
analyzed.
The products of PCR for RAPD analysis are visualized by Agarose Gel
Electrophoresis and after staining with Ethidium Bromide. The process of
RAPD does not involve any requirement of blotting or hybridization steps.
The DNA fragments obtained from RAPD can be further cloned and
sequenced to produce other types of markers like SNP.
7. Restriction Fragment Length Polymorphisms (RFLP) method of PCR-
mediated amplification is based on the fragments of genomic DNA previously
digested by restriction enzymes. It produces sets of molecular markers
necessary for animal or plant genome mapping project. The method is
capable of detecting the mutations occurring in each loci which manifests
by a gain or loss in the restriction sites. DNA obtained from homologous
chromosomes can be digested by restriction enzyme which shall yield
polymorphism in the DNA fragments obtained by electrophoresis.
RFLPs are sensitive and accurate PCR-based methods which can possibly
detect single nucleotide mutation, deletion or insertion within 1-100 bp of
DNA. This method, however, requires higher amount of DNA, and specific
probe libraries. The DNA patterns obtained may exhibit low polymorphism.
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 PCR Methods and
11.6 SUMMARY Primer Designing

• The Polymerase Chain Reaction (PCR) involves selective amplification of a

part of the DNA or RNA segment, specifically a gene fragment. PCR is a NOTES
widely applied method in molecular biology which enables gene cloning by
preparing multiple amplicons or copies of a particular gene.
• In molecular biology, an amplicon is a piece of DNA or RNA that is the
source and/or product of amplification or replication events. It can be formed
artificially, using various methods including Polymerase Chain Reactions
(PCR) or Ligase Chain Reactions (LCR), or naturally through gene
duplication.
• PCR has important applications in gene cloning, sequencing and target
detection.
• The process of DNA replication is catalyzed by the activity of Taq
polymerase, primers and dNTPs. The reaction carried out by the method
of PCR is accomplished by thermal cycles (heating and repeated cooling to
allow primer hybridization and replication).
• In 1976, the heat stable form of DNA polymerase was isolated from
thermophile Thermus aquaticus which can be stable at temperatures up to
95°C. Thermus aquaticus, is a species of bacteria that can tolerate high
temperatures. It is one of several thermophilic bacteria that belong to the
Deinococcus–Thermus group.
• The steps of PCR essentially comprises of 20-40 thermal cycles each of
which comprise of the steps carried out at specific temperatures and are
namely denaturation, annealing and extension.
• The PCR process was originally developed to amplify short segments of a
longer DNA molecule.
• A typical amplification reaction includes target DNA, a thermostable DNA
polymerase, two oligonucleotide primers, deoxynucleotide triphosphates
(dNTPs), reaction buffer and magnesium. Once assembled, the reaction is
placed in a thermal cycler, an instrument that subjects the reaction to a
series of different temperatures for set amounts of time.
• The primers are short nucleotide sequences complementary to the gene
sequence at the 5´ end. There are two sets of primers called forward primer
and reverse primer complementary to the sense and antisense strand of the
DNA, respectively.
• The deoxynucleotide triphosphate (dNTP) molecules are required in an
optimum concentration such that the reaction proceeds in a forward
equilibrium.
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PCR Methods and • The dNTPs function as building blocks for the synthesizing strand of DNA
Primer Designing
where they join by phosphor-diester linkage and form hydrogen bond with
the complementary base in the mother strand.
• Taq polymerase requires the presence of Mg2+ as a cofactor to maintain its
NOTES
catalytic activity.
• The steps of PCR essentially comprises of 20-40 thermal cycles each of
which comprise of the steps carried out at specific temperatures and are
namely denaturation, annealing and extension.
• The initial step of denaturation occurs at a higher temperature >90°C. Then
further cooling is done at a temperature of 4°C to store the amplicons.
• The temperature of each stage and the duration of reactions depend upon
various factors, such as the type of DNA polymerase used, nature of DNA
(GC content), melting temperature of primers, concentration of dNTPs and
cofactors in the reaction mixture.
• Specially engineered DNA polymerase called Hot Start Taq Polymerase
may require a higher denaturation temperature. The process of initial
denaturation helps in unwinding of the genomic DNA and preferably exposes
the gene segment accessible to the primer.
• The final hold stage at the end of final elongation occurs by lowering the
temperature to 4°C which helps in the storage of the reaction mixture. After
the accomplishment of around 30 cycles the amplicons are expected to
contain several copies of the desired gene sequence.
• To confirm the presence of amplicons resulting from replication of the desired
gene, the PCR products are resolved on an agarose gel electrophoresis
which results in the appearance of DNA band comparable with marker
DNA ladders.
• The gene amplicon should be represented by the desired size of band in
kilo base pairs (kbp). However, exact confirmation of the amplicons can
only be obtained by the process of gene sequencing.
• The success of obtaining correct amplicons of gene mainly depends upon
the choice of correct primers.
• The primers should essentially be of 17-30 nucleotides in length. The RAPD
primers should be around 10-12 nucleotide long.
• The nucleotide composition of the primer should comprise around 50%
GC content or 40-60%. The GC triple hydrogen bonds are stronger than
AT pairs thus increasing the melting point of the primer.
• The calculation of the melting point of the primer is regulated by the percentage
of AT and GC present in the primer. The formula is as follows,
Tm = 4°C x (Number of G’s and C’s in the Primer) + 2°C x (Number of
A’s and T’s in the Primer)
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• Both forward primer and reverse primer should have the similar Tm value. PCR Methods and
Primer Designing
Optimization of the primer to be selected for replication among a set of
primers should possesses the highest Tm.
• Primer sequences with more than 3 nucleotides repetitive in the sequence
NOTES
should be avoided. This results in the formation of secondary structure (hairpin
loop) which are undesirable for the PCR method.
• The forward primer and reverse primer should not contain any base
complementarities to each other.
• The number of GC bonds present at the 3´ end of the primer should not be
repetitively more than three. This would otherwise form non-specific
secondary structures or GC clamps.
• The online applications for primer designing usually require the DNA
sequences necessary to be referred to design the primers.
• The Primer-BLAST software and Primer3Plus software can be used as
these software helps in finding specific primers.
• Reverse Transcription PCR (RT-PCR) is type of PCR where initially a RNA
template is reverse transcribed into the complementary DNA catalyzed by
the activity of reverse transcriptase. The produced cDNA is amplified by
the help of PCR. Various sources like Avian Myeloblastosis Virus (AMV),
Moloney Murine Leukemia Virus (MMLV or MuLV) are implied to obtain
the enzyme reverse transcriptase.
• Real Time PCR (Quantitative PCR) method involves measuring the relative
amounts of target sequence in a sample. The amount of DNA generated
can be quantified by the assistance of a fluorescent probe. In this type of
PCR, DNA amplification is measured at the end of each cycle. The increasing
fluorescence intensity indicates the exponential increase in amplicons.
• Hot Start PCR involves mitigation of primer annealing or dimer formation.
The method involves withholding the reaction components like DNA
polymerase, PCR primers, dNTPs and cofactors from being involved in
reaction until the temperature of the first cycle reached to as high as 95°C,
i.e., higher than the annealing temperature.
• Inverse PCR amplification is possible with a single known internal sequence.
The primers are oriented in the reverse direction in respect to the normal
direction. The template in this type of reaction is a self-ligating restriction
fragment which forms a circle.
• Random Amplification of Polymorphic DNA (RAPD) method is variation
of PCR which develops molecular marker for establishing genetic maps or
to accomplish DNA finger printing.
• RAPD markers are of short length (10 nucleotides) and require a single
primer. The method does not require any prior information on the sequence
of DNA obtained from the tissue to be analyzed.
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PCR Methods and • The products of PCR for RAPD analysis are visualized by Agarose Gel
Primer Designing
Electrophoresis and after staining with Ethidium Bromide. The process of
RAPD does not involve any requirement of blotting or hybridization steps.
• The DNA fragments obtained from RAPD can be further cloned and
NOTES
sequenced to produce other types of markers like SNP.
• The RAPD analysis is a PCR-based method which involves amplification
of a target DNA in exponential forms. The primer used is arbitrary in nature
and involves usual PCR method of denaturation, annealing and elongation
in the presence of DNA polymerase and dNTPs.
• Restriction Fragment Length Polymorphisms (RFLP) method of PCR-
mediated amplification is based on the fragments of genomic DNA previously
digested by restriction enzymes. It produces sets of molecular markers
necessary for animal or plant genome mapping project. The method is
capable of detecting the mutations occurring in each loci which manifests
by a gain or loss in the restriction sites.
• DNA obtained from homologous chromosomes can be digested by
restriction enzyme which shall yield polymorphism in the DNA fragments
obtained by electrophoresis.
• RFLPs are sensitive and accurate PCR-based methods which can possibly
detect single nucleotide mutation, deletion or insertion within 1-100 bp of
DNA. This method, however, requires higher amount of DNA, and specific
probe libraries. The DNA patterns obtained may exhibit low polymorphism.

11.7 KEY WORDS

• Polymerase Chain Reaction (PCR): It involves selective amplification

of a part of the DNA or RNA segment, specifically a gene fragment; widely
applied method in molecular biology which enables gene cloning by
preparing multiple amplicons or copies of a particular gene.
• Amplicon: In molecular biology, an amplicon is a piece of DNA or RNA
that is the source and/or product of amplification or replication events.
• Thermus aquaticus: It is a species of bacteria that can tolerate high
temperatures, is one of several thermophilic bacteria that belong to the
Deinococcus–Thermus group.
• Primers: The primers are short nucleotide sequences complementary to
the gene sequence at the 5’ end, there are two sets of primers called forward
primer and reverse primer complementary to the sense and antisense strand
of the DNA, respectively.
• dNTPs: The deoxynucleotide triphosphate (dNTP) molecules are required
in an optimum concentration such that the reaction proceeds in a forward
equilibrium.
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 • Reaction buffer: It maintains the correct pH necessary for the reaction PCR Methods and
Primer Designing
conditions.
• Hot Start PCR: It involves mitigation of primer annealing or dimer formation.
• Inverse PCR amplification: It is possible with a single known internal NOTES
sequence, the primers are oriented in the reverse direction in respect to the
normal direction.
• Random Amplification of Polymorphic DNA (RAPD): This method is
variation of PCR which develops molecular marker for establishing genetic
maps or to accomplish DNA finger printing.
• Restriction Fragment Length Polymorphisms (RFLP): This method
of PCR-mediated amplification is based on the fragments of genomic DNA
previously digested by restriction enzymes.

11.8 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. Explain about the Polymerase Chain Reaction (PCR).
2. What are the components of PCR reaction procedure?
3. Name the steps involved in the PCR reaction.
4. Which procedure can be used for primer designing?
5. What is reverse transcription PCR?
6. Define real time PCR or the quantitative PCR method.
7. Explain the terms Hot Start PCR and Inverse PCR amplification.
8. Describe the Random Amplification of Polymorphic DNA (RAPD) method.
9. What is the significance of Restriction Fragment Length Polymorphisms
(RFLP) method?
Long Answer Questions
1. Briefly discuss about the Polymerase Chain Reaction (PCR) giving
appropriate examples.
2. Explain the components of PCR reaction procedure giving its specifications.
3. Discuss the steps involved in the PCR with the help of appropriate diagrams.
4. Briefly explain the terms denaturation, annealing, extension, final elongation
giving appropriate diagrams.
5. What are the advantages and limitations of Taq polymerase for being used
in PCR? Explain giving examples.
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PCR Methods and 6. Describe the software that helps in the primer designing procedure.
Primer Designing
7. Elaborate on reverse transcription PCR process and real time PCR
(quantitative PCR) method giving examples.
NOTES 8. Explain the significance and methodology involved in Hot Start PCR and
inverse PCR amplification.
9. Write detailed notes on the following:
(i) Random Amplification of Polymorphic DNA (RAPD) method
(ii) Restriction Fragment Length Polymorphisms (RFLP) method

11.9 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 DNA Sequencing

UNIT 12 DNA SEQUENCING Methods

METHODS
NOTES
Structure
12.0 Introduction
12.1 Objectives
12.2 Dna Sequencing: An Introduction
12.2.1 Sanger Sequencing
12.2.2 Sanger and Coulson Dideoxynucleotide Synthetic Method
12.2.3 Maxam–Gilbert’s Method of Sequencing
12.2.4 Automated Sequencing Method
12.2.5 DNA Microarray Analysis
12.3 Answers to Check Your Progress Questions
12.4 Summary
12.5 Key Words
12.6 Self Assessment Questions and Exercises
12.7 Further Readings

12.0 INTRODUCTION

DNA sequencing is the process of determining the nucleic acid sequence, i.e., the
order of nucleotides in DNA. It includes any method or technology that is used to
determine the order of the four bases - Adenine (A), Guanine (G), Cytosine (C),
and Thymine (T). Principally, the DNA sequencing is the determination of the
physical order of these bases in a molecule of DNA. However, there are many
other bases that may be present in a molecule. In some viruses, specifically the
bacteriophage, Cytosine (C) may be replaced by Hydroxy Methyl or Hydroxy
Methyl Glucose Cytosine. In mammalian DNA, variant bases with methyl groups
or phosphor-sulfate may be found.
Sanger sequencing is a method of DNA sequencing and is based on the
selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase
during in vitro DNA replication. It was developed by British biochemist Frederick
Sanger and colleagues in 1977. In Sanger sequencing, the target DNA is copied
many times, making fragments of different lengths. Fluorescent ‘chain terminator’
nucleotides mark the ends of the fragments and allow the sequence to be
determined. Regions of DNA up to about 900 base pairs (bp) in length are routinely
sequenced using the Sanger sequencing or the chain-termination method.
Maxam–Gilbert sequencing is a method of DNA sequencing developed by
Allan Maxam and Walter Gilbert in 1976–1977. This method is based on
nucleobase-specific partial chemical modification of DNA and subsequent cleavage
of the DNA backbone at sites adjacent to the modified nucleotides. Maxam–
Gilbert sequencing was the first widely adopted method for DNA sequencing and
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DNA Sequencing along with the Sanger dideoxy method, represents the first generation of DNA
Methods
sequencing methods. This sequencing is succeeded by Next-Generation sequencing
methods. Sanger–Coulson sequencing is chain-termination method. It uses single-
stranded (ss) DNA which is usually cloned in M13 phage vector. DNA microarrays
NOTES revolutionized the approach to gene expression profiling. Compared to prior
methods, DNA microarrays have theatrically high throughput and are less
cumbersome. Microarrays developed as a technique for large-scale DNA mapping
and sequencing. Knowledge of DNA sequences has become indispensable for
biological research, medical diagnosis, biotechnology, forensic biology, virology
and biological systematics.
In this unit, you will study about the DNA sequencing, Sanger sequencing,
Sanger and Coulson dideoxynucleotide synthetic method, Maxam–Gilbert’s method
of sequencing, automated sequencing method, and DNA microarray analysis.

12.1 OBJECTIVES

After going through this unit, you will be able to:

• Understand the significance of DNA sequencing
• Explain the various methods of DNA sequencing
• Discuss the Sanger and Coulson dideoxynucleotide synthetic method and
its components
• Define the Maxam–Gilbert’s method of sequencing
• Elucidate on automated sequencing method
• Explain the method and applications of DNA microarray analysis

12.2 DNA SEQUENCING: AN INTRODUCTION

DNA sequencing is the process of determining the nucleic acid sequence – the
order of nucleotides in DNA. It includes any method or technology that is used to
determine the order of the four bases - Adenine (A), Guanine (G), Cytosine (C),
and Thymine (T). Principally, the DNA sequencing is the determination of the
physical order of these bases in a molecule of DNA. However, there are many
other bases that may be present in a molecule. In some viruses, specifically the
bacteriophage, Cytosine (C) may be replaced by Hydroxy Methyl or Hydroxy
Methyl Glucose Cytosine. In mammalian DNA, variant bases with methyl groups
or phosphor-sulfate may be found. Depending on the sequencing technique, a
particular modification, for example, the 5mC (5 methyl Cytosine) common in
humans, may or may not be detected.
DNA sequencing can be used for determining the sequence of individual
genes, larger genetic regions, i.e., clusters of genes or operons, full chromosomes
or entire genomes of any organism. Additionally, the DNA sequencing is considered
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as the most efficient method to indirectly sequence RNA or proteins. The sequencing DNA Sequencing
Methods
attained with DNA sequencing technology helps in the sequencing of complete
DNA sequences, or genomes of numerous types and species of life, including the
human genome and other complete DNA sequences of many animal, plant, and
microbial species. NOTES
The first DNA sequences were obtained in the early 1970s by academic
researchers using laborious methods based on two-dimensional chromatography.
Following the development of fluorescence-based sequencing methods with a DNA
sequencer the DNA sequencing has become easier.
Sanger sequencing is a method of DNA sequencing and is based on the
selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase
during in vitro DNA replication. It was developed by British biochemist Frederick
Sanger and colleagues in 1977. In Sanger sequencing, the target DNA is copied
many times, making fragments of different lengths. Fluorescent ‘chain terminator’
nucleotides mark the ends of the fragments and allow the sequence to be
determined. Regions of DNA up to about 900 base pairs (bp) in length are routinely
sequenced using the Sanger sequencing or the chain-termination method.
The conventional methods of DNA sequencing were used in the early 1990s
which involved radioactive dideoxy bases coupled to termination reaction or reagent
based degradation method. However, along with advancements in micro-technology
and automation, high throughput gene sequencers have replaced the old methods.
The conventional methods of DNA sequencing possessed limitations like use of
hazardous chemicals, analysis of short DNA sequence around 250 bp. Automated
DNA sequencing involves application of fluorescence method for deciphering DNA
sequencing. This method reduces the need of tedious multi-lane gels used in old
methods. DNA microarray analysis enables the detection and expression analysis
of numerous genes together.
The method adopted by Sanger (1974) involves the application of the
principle of DNA replication necessary for the dideoxy sequencing method. The
chain-termination reaction involved in the process was accomplished by the use of
dideoxyribose sugars in which the hydroxyl group is absent at both 2´ and 3´ position
of the Carbon atom. The amount of DNA preferably sequenced by this method
usually ranges up to 800-900 base pairs in length. The human genome project has
been accomplished by applying this technique of DNA sequencing to decipher
smaller fragments of the human DNA. Maxam–Gilbert’s method of sequencing
was reported by Maxam and Gilbert (1977) which is commonly termed as the
chemical degradation method. This method is different from that of Sanger’s method
in the fact that the double stranded DNA desired to be sequenced is radiolabelled
at the 3´ end. The method of automated sequencing utilizes the fluorescent dye to
label the nucleotides. The fluorescent dye is non-hazardous in comparison to
radioisotopes. The fluorescence intensity is analyzed by laser mediated detection.
With advancements in the technical aspects there has been no requirement of
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DNA Sequencing adding the samples into the gel lane. Fluorescent labels are integrated into the
Methods
DNA strands by using 5´-labelled primers or 3´ labelled dideoxynucleotide
triphosphates which functions as terminators. The labelling at the 3´ end by using
dideoxyNTP enables the use of different fluorescent dyes to label the bases. The
NOTES different fluorochromes implied are likely to exhibit different wavelengths of emission
upon excitation byArgon Laser. The method of DNA or nucleic acid array enables
immobilization of DNA to a surface facilitated by covalent or non-covalent
interactions. The fluorescent probes are utilized to bind with the specific nucleic
acid sequences in the immobilized DNA material. Automation has enabled the
direct synthesis of DNA segments on the surface of immobilization plate where
the probes are added to detect the presence of the gene. The automated self
assembled arrays help to provide repeatability in the experiments and also helps
to analyze a wide number of samples. The primary application of microarray method
involves analysis of gene expression. The extracted RNA obtained from the cells
is converted into cDNA or cRNA. The allelic variations are successfully analysed
by the method of microarray analysis which indicates the differences among various
genotypes.
12.2.1 Sanger Sequencing
Sanger sequencing is a method of DNA sequencing and is based on the selective
incorporation of chain-terminating dideoxynucleotides by DNA polymerase during
in vitro DNA replication. Developed by Frederick Sanger and colleagues in 1977,
it was the most widely used sequencing method for approximately 40 years. More
recently, higher volume Sanger sequencing has been replaced by ‘Next-Gen’ or
Next-Generation sequencing methods, especially for large scale, automated genome
analyses.
Fundamentally, in the Sanger sequencing, the target DNA is copied many
times, making fragments of different lengths. Fluorescent ‘chain terminator’
nucleotides mark the ends of the fragments and allow the sequence to be
determined.
Next-Generation sequencing techniques are new, use the large scale
approaches that increase the speed and reduce the cost of DNA sequencing.
However, the Sanger method remains in wide use, for smaller scale projects, and
for validation of Next-Gen results.
Sanger Sequencing: The Chain-Termination Method
Regions of DNA up to about 900 base pairs in length are routinely sequenced
using a method called Sanger sequencing or the chain-termination method.
In the development of Human Genome, Sanger sequencing was used for
determining the sequences of many relatively small fragments of human DNA. The
fragments were aligned based on overlapping portions to assemble the sequences
of larger regions of DNA and, ultimately the entire chromosomes. Sanger
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sequencing is widely used for the sequencing of individual pieces of DNA, such as DNA Sequencing
Methods
fragments used in DNA Cloning or generated through the Polymerase Chain
Reaction (PCR).
Components for Sanger Sequencing NOTES
Sanger sequencing can make many copies of a target DNA region. Its components
are similar to those needed for DNA replication in an organism, or for Polymerase
Chain Reaction (PCR), which copies DNA in vitro. The components include the
following:
• A DNA polymerase enzyme.
• A Primer, which is a short piece of single-stranded (ss) DNA that binds to
the template DNA and acts as a ‘Starter’ for the polymerase.
• The four DNA nucleotides – dATP, dTTP, dCTP, dGTP.
• The template DNA to be sequenced.
• Dideoxy, or Chain-Terminating, versions of all four nucleotides (ddATP,
ddTTP, ddCTP, ddGTP), each labelled with a different colour of dye.
Figure 12.1 illustrates the structure of Whole-Genome Sequencing.

Fig. 12.1 Structure of Whole-Genome Sequencing

Consequently, the classical chain-termination method requires a single-stranded

(ss) DNA template, a DNA primer, a DNA polymerase, normal deoxynucleo-
tidetriphosphates (dNTPs), and modified di-deoxynucleotidetriphosphates
(ddNTPs), the latter of which terminate DNA strand elongation. These chain-
terminating nucleotides lack a 3'-OH group required for the formation of a phosphor-
diester bond between two nucleotides, causing DNA polymerase to cease extension
of DNA when a modified ddNTP is incorporated. The ddNTPs may be radioactively
or fluorescently labelled for detection in automated sequencing machines.
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DNA Sequencing Dideoxy nucleotides are similar to regular or deoxy nucleotides, but there is
Methods
one key difference, i.e., they lack a hydroxyl group on the 3´ Carbon of the Sugar
Ring. In a regular nucleotide, the 3´ hydroxyl group allows a new nucleotide to be
added to an existing chain.
NOTES
Once a dideoxy nucleotide has been added to the chain, there is no hydroxyl
available and no further nucleotides can be added. The chain ends with the dideoxy
nucleotide, which is marked with a particular colour of dye depending on the base
(A, T, C or G) that it carries.
Method of Sanger Sequencing
The DNA sample is divided into four separate sequencing reactions, containing all
four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the
DNA polymerase. To each reaction is added only one of the four
dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP), while the other added
nucleotides are ordinary ones. The dideoxynucleotide concentration should be
approximately 100-fold lower than that of the corresponding deoxynucleotide
(for example, 0.005mM ddTTP : 0.5mM dTTP) to allow enough fragments to be
produced while still transcribing the complete sequence. Four separate reactions
are required in this process to test all four ddNTPs. Subsequent rounds of template
DNA extension from the bound primer, the resulting DNA fragments are heat
denatured and separated by size using gel electrophoresis.
In the original publication of 1977, the formation of base paired loops of
ssDNA was difficult in resolving bands at some locations. This is often performed
using a denaturing Polyacrylamide-Urea Gel with each of the four reactions running
in one of four individual lanes of A, T, G, C. The DNA bands may then be visualized
by autoradiography or UV light and the DNA sequence can be directly read off
the X-ray film or gel image.
Technical variations of chain-termination sequencing include tagging with
nucleotides containing radioactive phosphorus for radiolabelling, or using a primer
labelled at the 5' end with a fluorescent dye. Dye-primer sequencing facilitates
reading in an optical system for faster and more economical analysis and automation.
12.2.2 Sanger and Coulson Dideoxynucleotide Synthetic Method
The method adopted by Sanger (1974) involves the application of the principle of
DNA replication necessary for the dideoxy sequencing method. The 3´ hydroxyl
group is necessary to develop the chain-termination reaction. The chain-termination
reaction involved in the process was accomplished by the use of deoxyribose
sugars in which the hydroxyl group is absent at both 2´ and 3´ position of the
Carbon atom. This results in the termination of DNA chain elongation resulting
due to inability in the formation of otifs r-diester bond with adjacent
deoxynucleotide. In the chain-termination reaction the synthesis of the second
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strand is dependent on the single stranded DNA template. The process of obtaining DNA Sequencing
Methods
a single stranded template DNA is usually performed by cloning the DNA in M13
vector or by NaOH digestion. The complementary strand is synthesized by an
enzymatic process. The amount of DNA preferably sequenced by this method
usually ranges up to 800-900 base pairs in length. The human genome project has NOTES
been accomplished by applying this technique of DNA sequencing to decipher
smaller fragments of the human DNA. Shotgun sequencing enables to align the
overlapping ends of larger fragments of DNA within a chromosome. Various
automated sequencing methods have replaced this old conventional method of
DNA sequencing. However, this method still finds its application in sequencing
individual pieces of DNA fragments in cloning or PCR.
Components of Sanger and Coulson Dideoxynucleotide Synthetic Method
Following are the components of the Sanger and Coulson dideoxynucleotide
synthetic method.
1. Annealing with Primer: The initial stage of the sequencing method involves
binding of a suitable primer to a recombinant M13 vector containing DNA
or separately to a single stranded DNA. The process of DNA polymerase
mediated synthesis of complementary strand initiates with the use of the
primer. Interestingly, the primer is radiolabelled which shall facilitate to obtain
a radiograph signal of the DNA strand being synthesized. The reaction mixture
may also contain radiolabelled deoxynucleotides like S35 or P32. Figure 12.2
illustrates the annealing of primer to template DNA.

Fig. 12.2 Annealing of Primer to Template DNA

2. Activity of the Enzyme: The enzyme used in the method of Sanger’s

sequencing lacks 3´-5´ exonuclease activity. This feature facilitates to
generate a DNA ladder. Klenow fragment was earlier implied to accomplish
DNA synthesis. DNA polymerase adds deoxynucleotide or the corresponding
2´, 3´ dideoxynucleotide during the extension step. Current advancements
have implied the use sequenase which is a T7 Bacteriophage DNA
Polymerase. This enzyme has replaced the use of Klenow fragment. The
efficiency of sequenase has been observed to be higher than Klenow
fragment. Among various sequencing polymerases, the sequenase has been
reported to incorporate ddNTPs with 33% efficiency. The enzyme is a form
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DNA Sequencing of modified DNA polymerase which can easily catalyze the extension of
Methods
DNA across GC sequences.
3. Synthesis of Complementary Strand: The strand synthesis occurs in
NOTES presence of the reaction mixture which contains the polymerase enzyme,
single stranded template DNA and 5´ radiolabelled DNA primers, mixture
of dNTP with dideoxydNTP form and the remaining three dNTPs. The
mixture involves four parallel reactions occurring in the presence of different
dideoxynucleotide, i.e., dideoxyATP, dideoxyTTP, dideoyGTP, dideoxyCTP.
The concentration of dideoxyNTP should preferably only 1% of the particular
dNTP. This enables to obtain a series of labelled strands being terminated
by dideoxy NTP. The length of the radio-labelled strand depends upon the
position of the dideoxy NTP at the 5´ end. Figure 12.3 illustrates the synthesis
of nascent (daughter) strand complementary to template strand.

Fig. 12.3 Synthesis of Nascent Strand Complementary to Template Strand

4. Autoradiographic Analysis of DNA Sequence: PolyAcrylamide Gel

Electrophoretic (PAGE) separation is performed. Separate reaction mixture
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is loaded into each lanes of the gel. The reaction mixtures are prepared so DNA Sequencing
Methods
as to differentiate the strands on the basis of a single nucleotide difference.
The pore size in polyacrylamide gels is smaller than that of agarose gels.
The gels used for separation of strands are denaturing in nature which are NOTES
prepared by addition of Urea or Formamide. Urea and Formamide are
capable of lowering the melting temperature of DNA molecules. The
denaturants separate the parent strand and thus separates the newly
synthesized strand. A higher voltage is preferred to prevent renaturation of
the DNA strands. The gels are washed in distilled water and the DNA
bands are transferred to a nitrocellulose membrane by the help of blotting
method. The nitrocellulose membranes are incubated for autoradiography
so that the bands having 5´ radiolabelled molecules are only detected. The
strands are separated on the gel on the basis of their size. The smallest
single dideoxynucleotide which terminated the process of chain elongation
at the first stage appears at the base of the gel. This nucleotide thus represents
the first sequence of the DNA strand. The sequential sequences are read
from the bottom to up approach. Figure 12.4 illustrates the strand synthesis
associated with radioactive dideoxyNTPs while Figure 12.5 illustrates the
PAGE mediate separation of DNA strands and sequencing reading from
bottom to top.

Fig. 12.4 Strand Synthesis Associated with Radioactive DideoxyNTPs

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DNA Sequencing
Methods

NOTES

Fig. 12.5 PAGE Mediate Separation of DNA Strands and

Sequencing Reading from Bottom to Top

12.2.3 Maxam–Gilbert’s Method of Sequencing

Maxam–Gilbert sequencing is a method of DNA sequencing developed by Allan
Maxam and Walter Gilbert in 1976–1977. This method is based on nucleobase-
specific partial chemical modification or the chemical degradation method of DNA
and subsequent cleavage of the DNA backbone at sites adjacent to the modified
nucleotides. This method is different from that of Sanger’s method in the fact that
the double stranded DNA desired to be sequenced is radiolabelled at the 3´ end.
The DNA strand is selectively fragmented for the breakdown of specific base
pairs. The fragmented DNA strands are loaded into the polyacrylamide gel. The
strands get separated on the basis of their size. The bands are detected by
autoradiography. The method is as follows:
1. The DNA is fragmented by restriction endonuclease and labelled with
radiolabelled Phosphates. The radiolabelling is performed by the activity of
the enzyme Polynucleotide Kinase. The labelled strands are separated with
radioactive labelling at the 5´ end.
2. The labelled DNA strands are segregated into four reaction mixtures each
of which is treated with different reagents which can specifically cleave G,
C, AT or GC nucleotides in the DNA fragments. The concentration of the
reagents is adjusted such that it degrades 50% of the bases.
3. The fragments of different sizes are produced due to degradation caused
by the reagents.
4. The different samples are loaded into different lanes of Polyacrylamide Gel.
The fragments are separated by electrophoresis based on their size.
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 5. The sequences are detected on the basis of the auto-radiographed fragments DNA Sequencing
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in each lane.
Figure 12.6 illustrates the Maxam–Gilbert method of sequencing of DNA.
NOTES

Fig. 12.6 Maxam–Gilbert Method of Sequencing of DNA

12.2.4 Automated Sequencing Method

The method of automated sequencing utilizes the fluorescent dye to label the
nucleotides. The fluorescent dye is non-hazardous in comparison to radioisotopes.
The fluorescence intensity is analyzed by laser mediated detection. The fluorescent
emission is received by a charge coupled device that determines the wavelength.
The automated sequencers are designed to detect different wavelengths of
fluorescent dyes implied to label the four bases. This enables to detect the sequential
identification of the bases in DNA fragment. In older systems of automated DNA
sequencing, the DNA sample after being labelled was loaded in a single lane of the
gel. However, with advancements in the technical aspects there has been no
requirement of adding the samples into the gel lane. The sequencer can perform
the labelling and analysis. Alternately, the gel containing the labelled DNA bases
can be placed in a DNA sequencer to detect the bases. The method is more
efficient than other conventional methods. The features of the automated DNA
sequencing is as follows.
1. Fluorescent labels are integrated into the DNA strands by using 5´-labelled
primers or 3´ labelled Dideoxynucleotide Triphosphates which functions as
terminators. The labelling at the 3´ end by using DideoxyNTP enables the
use of different Fluorescent Dyes to label the bases.
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DNA Sequencing 2. The reaction is performed in a single test tube by performing co-incubation
Methods
of Fluorochromes.
3. The different Fluorochromes implied are likely to exhibit different
wavelengths of emission upon excitation by Argon Laser.
NOTES
4. The DNA fragments are analyzed by separation in a single lane of a gel or
by performing capillary gel electrophoresis.
5. Capillaries used in this method have diameter of 50 mm and possesses high
surface area to volume ratio. This enables dissipation of heat energy.
6. Capillary method is highly automated and controlled. The process can be
performed at a much higher voltage, thus consuming very less time.

A.

B.

Fig. 12.7 (A) Separation of DNA (Labelled) by Capillary Electrophoresis, and

(B) Detection of Fluorescence by Detector

In the Figure 12.7, (A) illustrates the separation of DNA (labelled) by capillary
electrophoresis, and (B) illustrates the detection of fluorescence by detector.
Different colour codes for the four bases exhibited due to different emission
wavelengths of the Fluorochromes.
12.2.5 DNA Microarray Analysis
The method of DNA or nucleic acid array enables immobilization of DNA to a
surface facilitated by covalent or non-covalent interactions. The fluorescent probes
are utilized to bind with the specific nucleic acid sequences in the immobilized
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DNA material. The fluorescent array indicates the qualitative analysis of the genes. DNA Sequencing
Methods
Around 25-60 bp long oligo fragments of DNA can be detected by fluorescent
based microarrays. The array method used can be of three types namely,
1. Glass Spotted Arrays
NOTES
2. In Situ Synthesized Arrays
3. Self-Assembled Arrays
Poly-Lysine coated glass slides are used for the binding of DNA materials and are
suitable for detection by Fluorescent Array. Fluorescent detection method provides
various advantages over the conventional radioactive or chemiluminescence
methods. The Fluorescent method is quite specific and non-hazardous. Automation
has enabled the direct synthesis of DNA segments on the surface of immobilization
plate where the probes are added to detect the presence of the gene. In conventional
methods the synthesized DNA fragments are added for array detection.
Interestingly in the automated self assembled arrays, the DNA is synthesized on
the Polystyrene Beads. The beads are encoded with Fluorescent probes which
detect the presence of a particular DNA sequence. This is followed by optical
decoding of the Fluorescent signals which enables to obtain the qualitative analysis.
The automated self assembled arrays help to provide repeatability in the experiments
and also helps to analyze a wide number of samples.
Method of DNA Microarray Analysis
1. The presence of gene chips which contain microscopic DNA spots prepared
by immobilization of DNA on the surface.
2. The method of microarray detection enables to undertake quantitative analysis
of a larger number of genes simultaneously.
3. The spots of DNA contain Pico-moles of DNA sequence which functions
as specific probes or reporter sequence. The probes are complementary to
cDNA sample obtained from the target tissue.
4. The probe binds to the target sequence which results in the emission of
Fluorescence obtained from the Fluorochromes.
5. The probes are synthesized and bound to solid surface composed of Epoxy-
Silane, Amino-Silane, Lysine or Polyacrylamide. The probe binds to the
surface by the virtue of covalent bonding. The solid surface implied for
binding of DNA is termed as Micro Gene Chip.
6. The strength of hybridization depends upon the amount of target sequence
resent for binding with the probe sequence. Correspondingly, the intensity
of Fluorescent signal indicates the amount of target sequence present in the
chips.
7. The similar spots obtained from different analyte tissue are compared for
spot intensity and thus indicate levels of gene expression among various
samples.
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DNA Sequencing Application of DNA Microarray Analysis
Methods
1. Analysis of Gene Expression: The primary application of microarray
method involves analysis of gene expression. The extracted RNA obtained
NOTES from the cells is converted into cDNA or cRNA. The cDNA or cRNA is
preferably synthesized by incorporating Fluorescent or Biotin labelled
Nucleotides. Followed by hybridization of the cDNA/cRNA in the chip it is
incubated with Fluorescent probe or libelled Streptavidin.
2. Analysis of Transcription Factor Binding Assay: The method of
microarray analysis is coupled to Chromatin Immuno-Precipitation which
determines the binding sites of transcription factors. The signal of the probe-
DNA binding is regulated by the magnitude of transcription factors binding
to the specific DNA motifs.
3. Genotype Analysis: The allelic variations are successfully analysed by the
method of microarray analysis which indicates the differences among various
genotypes. However, allelic hybridization may result in background problems
arising from non-specific binding.
Figure 12.8 illustrates the method of microarray analysis.

Fig. 12.8 Method of Microarray Analysis

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 DNA Sequencing
Methods
Check Your Progress
1. What is DNA sequencing?
2. Why DNA sequencing is used? NOTES
3. Explain Sanger sequencing.
4. How Sanger sequencing method helps in DNA sequencing?
5. Define Sanger and Coulson dideoxynucleotide synthetic method.
6. What is Maxam–Gilbert sequencing?
7. What does the method of automated sequencing utilizes?

12.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. DNA sequencing is the process of determining the nucleic acid sequence –

the order of nucleotides in DNA. It includes any method or technology that
is used to determine the order of the four bases - Adenine (A), Guanine
(G), Cytosine (C), and Thymine (T). Principally, the DNA sequencing is the
determination of the physical order of these bases in a molecule of DNA.
2. DNA sequencing can be used for determining the sequence of individual
genes, larger genetic regions, i.e., clusters of genes or operons, full
chromosomes or entire genomes of any organism. Additionally, the DNA
sequencing is considered as the most efficient method to indirectly sequence
RNA or proteins.
The sequencing attained with DNA sequencing technology helps in the
sequencing of complete DNA sequences, or genomes of numerous types
and species of life, including the human genome and other complete DNA
sequences of many animal, plant, and microbial species.
3. The Sanger sequencing is a method of DNA sequencing and is based on
the selective incorporation of chain-terminating dideoxynucleotides by DNA
polymerase during in vitro DNA replication. It was developed by Frederick
Sanger and colleagues in 1977. Fundamentally, in the Sanger sequencing,
the target DNA is copied many times, making fragments of different lengths.
Fluorescent ‘chain terminator’ nucleotides mark the ends of the fragments
and allow the sequence to be determined.
4. The DNA sample is divided into four separate sequencing reactions,
containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP
and dTTP) and the DNA polymerase. To each reaction is added only one
of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP), while
the other added nucleotides are ordinary ones. The dideoxynucleotide
concentration should be approximately 100-fold lower than that of the
corresponding deoxynucleotide to allow enough fragments to be produced
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DNA Sequencing while still transcribing the complete sequence. The formation of base paired
Methods
loops of ssDNA was difficult in resolving bands at some locations. This is
often performed using a denaturing Polyacrylamide-Urea Gel with each of
the four reactions running in one of four individual lanes of A, T, G, C. The
NOTES DNA bands may then be visualized by autoradiography or UV light and the
DNA sequence can be directly read off the X-ray film or gel image.
5. The Sanger and Coulson dideoxynucleotide synthetic method was adopted
by Sanger (1974). It involves the application of the principle of DNA
replication necessary for the dideoxy sequencing method. The 3´ hydroxyl
group is necessary to develop the chain-termination reaction. The chain-
termination reaction involved in the process was accomplished by the use
of deoxyribose sugars in which the hydroxyl group is absent at both 2´ and
3´ position of the Carbon atom. This results in the termination of DNA
chain elongation resulting due to inability in the formation of otifs r-diester
bond with adjacent deoxynucleotide.
6. Maxam–Gilbert sequencing is a method of DNA sequencing developed by
Allan Maxam and Walter Gilbert in 1976–1977. This method is based on
nucleobase-specific partial chemical modification or the chemical
degradation method of DNA and subsequent cleavage of the DNA
backbone at sites adjacent to the modified nucleotides. The DNA strand is
selectively fragmented for the breakdown of specific base pairs. The
fragmented DNA strands are loaded into the polyacrylamide gel. The strands
get separated on the basis of their size. The bands are detected by
autoradiography.
7. The method of automated sequencing utilizes the fluorescent dye to label
the nucleotides. The fluorescent dye is non-hazardous in comparison to
radioisotopes. The fluorescence intensity is analyzed by laser mediated
detection. The fluorescent emission is received by a charge coupled device
that determines the wavelength. The automated sequencers are designed to
detect different wavelengths of fluorescent dyes implied to label the four
bases. This enables to detect the sequential identification of the bases in
DNA fragment.

12.4 SUMMARY

• DNA sequencing is the process of determining the nucleic acid sequence –

the order of nucleotides in DNA. It includes any method or technology that
is used to determine the order of the four bases - Adenine (A), Guanine
(G), Cytosine (C), and Thymine (T).
• Principally, the DNA sequencing is the determination of the physical order
of these bases in a molecule of DNA. However, there are many other bases
that may be present in a molecule.
• In some viruses, specifically the bacteriophage, Cytosine (C) may be
replaced by Hydroxy Methyl or Hydroxy Methyl Glucose Cytosine.
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• In mammalian DNA, variant bases with methyl groups or phosphor-sulfate DNA Sequencing
Methods
may be found. Depending on the sequencing technique, a particular
modification, for example, the 5mC (5 methyl Cytosine) common in humans,
may or may not be detected.
• DNA sequencing can be used for determining the sequence of individual NOTES
genes, larger genetic regions, i.e., clusters of genes or operons, full
chromosomes or entire genomes of any organism. Additionally, the DNA
sequencing is considered as the most efficient method to indirectly sequence
RNA or proteins.
• The sequencing attained with DNA sequencing technology helps in the
sequencing of complete DNA sequences, or genomes of numerous types
and species of life, including the human genome and other complete DNA
sequences of many animal, plant, and microbial species.
• Sanger sequencing is a method of DNA sequencing and is based on the
selective incorporation of chain-terminating dideoxynucleotides by DNA
polymerase during in vitro DNA replication. Developed by Frederick Sanger
and colleagues in 1977, it was the most widely used sequencing method for
approximately 40 years.
• More recently, higher volume Sanger sequencing has been replaced by
‘Next-Gen’ or Next-Generation sequencing methods, especially for large
scale, automated genome analyses.
• Fundamentally, in the Sanger sequencing, the target DNA is copied many
times, making fragments of different lengths. Fluorescent ‘chain terminator’
nucleotides mark the ends of the fragments and allow the sequence to be
determined.
• Next-Generation sequencing techniques are new, use the large scale
approaches that increase the speed and reduce the cost of DNA sequencing.
However, the Sanger method remains in wide use, for smaller scale projects,
and for validation of Next-Gen results.
• Regions of DNA up to about 900 base pairs in length are routinely
sequenced using a method called Sanger sequencing or the chain-termination
method.
• Sanger sequencing is widely used for the sequencing of individual pieces of
DNA, such as fragments used in DNA Cloning or generated through the
Polymerase Chain Reaction (PCR).
• Sanger sequencing can make many copies of a target DNA region. Its
components are similar to those needed for DNA replication in an organism,
or for Polymerase Chain Reaction (PCR), which copies DNA in vitro.
• Consequently, the classical chain-termination method requires a single-
stranded (ss) DNA template, a DNA primer, a DNA polymerase, normal
deoxynucleotidetriphosphates (dNTPs), and modified di-deoxynucleo-
tidetriphosphates (ddNTPs), the latter of which terminate DNA strand
elongation.
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DNA Sequencing • Dideoxy nucleotides are similar to regular or deoxy nucleotides, but there is
Methods
one key difference, i.e., they lack a hydroxyl group on the 3´ Carbon of the
Sugar Ring. In a regular nucleotide, the 3´ hydroxyl group allows a new
nucleotide to be added to an existing chain.
NOTES • Once a dideoxy nucleotide has been added to the chain, there is no hydroxyl
available and no further nucleotides can be added. The chain ends with the
dideoxy nucleotide, which is marked with a particular colour of dye
depending on the base (A, T, C or G) that it carries.
• The DNA sample is divided into four separate sequencing reactions,
containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP
and dTTP) and the DNA polymerase. To each reaction is added only one
of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP), while
the other added nucleotides are ordinary ones.
• In the original publication of 1977, the formation of base paired loops of
ssDNA was difficult in resolving bands at some locations. This is often
performed using a denaturing Polyacrylamide-Urea Gel with each of the
four reactions running in one of four individual lanes of A, T, G, C.
• The DNA bands can be visualized by autoradiography or UV light and the
DNA sequence can be directly read off the X-ray film or gel image.
• Sanger and Coulson dideoxynucleotide synthetic method adopted by Sanger
(1974) involves the application of the principle of DNA replication necessary
for the dideoxy sequencing method.
• The 3´ hydroxyl group is necessary to develop the chain-termination reaction.
The chain-termination reaction involved in the process was accomplished
by the use of deoxyribose sugars in which the hydroxyl group is absent at
both 2´ and 3´ position of the Carbon atom.
• The process of obtaining a single stranded template DNA is usually performed
by cloning the DNA in M13 vector or by NaOH digestion.
• Maxam–Gilbert sequencing is a method of DNA sequencing developed by
Allan Maxam and Walter Gilbert in 1976–1977. This method is based on
nucleobase-specific partial chemical modification or the chemical
degradation method of DNA and subsequent cleavage of the DNA
backbone at sites adjacent to the modified nucleotides.
• The DNA strand is selectively fragmented for the breakdown of specific
base pairs. The fragmented DNA strands are loaded into the polyacrylamide
gel. The strands get separated on the basis of their size. The bands are
detected by autoradiography.
• The method of automated sequencing utilizes the fluorescent dye to label
the nucleotides. The fluorescent dye is non-hazardous in comparison to
radioisotopes.
• The fluorescence intensity is analyzed by laser mediated detection. The
fluorescent emission is received by a charge coupled device that determines
the wavelength.
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 • The automated sequencers are designed to detect different wavelengths of DNA Sequencing
Methods
fluorescent dyes implied to label the four bases. This enables to detect the
sequential identification of the bases in DNA fragment.
• The method of DNA or nucleic acid array enables immobilization of DNA
to a surface facilitated by covalent or non-covalent interactions. NOTES
• The fluorescent probes are utilized to bind with the specific nucleic acid
sequences in the immobilized DNA material. The fluorescent array indicates
the qualitative analysis of the genes. Around 25-60 bp long oligo fragments
of DNA can be detected by fluorescent based microarrays.

12.5 KEY WORDS

• DNA sequencing: It is the process of determining the nucleic acid sequence

– the order of nucleotides in DNA. It includes any method or technology
that is used to determine the order of the four bases - Adenine (A), Guanine
(G), Cytosine (C), and Thymine (T).
• Sanger sequencing: This method of DNA sequencing was developed by
Frederick Sanger and colleagues in 1977, and is based on the selective
incorporation of chain-terminating dideoxynucleotides by DNA polymerase
during in vitro DNA replication.
• Maxam–Gilbert sequencing: This method of DNA sequencing was
developed by Allan Maxam and Walter Gilbert in 1976–1977, and is based
on nucleobase-specific partial chemical modification or the chemical
degradation method of DNA and subsequent cleavage of the DNA
backbone at sites adjacent to the modified nucleotides.

12.6 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. Explain DNA sequencing giving example.
2. What does DNA sequencing determine?
3. What is Sanger sequencing?
4. Explain the chain-termination method of Sanger sequencing.
5. What are the components of Sanger sequencing?
6. State the Maxam–Gilbert’s method of sequencing.
7. Explain the automated DNA sequencing method.
8. What is DNA microarray analysis?
9. List the applications of DNA microarray analysis.
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DNA Sequencing Long Answer Questions
Methods
1. Briefly discuss about the DNA Sequencing method and its significance in
DNA analysis and medical sciences.
NOTES 2. Discuss the characteristic features and methods of Sanger sequencing giving
appropriate examples.
3. Diagrammatically explain the principle and methods of Sanger’s dideoxy
method of DNA sequencing? Why is it called a chain-termination method?
Explain.
4. Describe the Sanger and Coulson dideoxynucleotide synthetic method and
components required for DNA sequencing.
5. Brief on the Maxam–Gilbert’s method of sequencing. Also compare the
Sanger’s method and Maxam–Gilbert’s method of DNA sequencing. Why
is the later called as degradation method?
6. What do you mean by automated DNA sequencing? Explain the method of
fluorescence detection of DNA sequences.
7. Explain the principle, method and applications of DNA microarray analysis.
8. What are gene chips? Explain about the self-assembled microarray method
with the help of examples.

12.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
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 Gene Silencing and

UNIT 13 GENE SILENCING AND Antisense Technology

ANTISENSE TECHNOLOGY
NOTES
Structure
13.0 Introduction
13.1 Objectives
13.2 Gene Silencing: An Introduction
13.2.1 Transcriptional Gene Silencing (TGS)
13.2.2 Genetic Factors Associated with Transcriptional Gene Silencing (TGS)
13.2.3 Post-Transcriptional Gene Silencing (PTGS)
13.3 Formation of Antisense mRNA and Inhibition of Gene Expression
13.4 Answers to Check Your Progress Questions
13.5 Summary
13.6 Key Words
13.7 Self Assessment Questions and Exercises
13.8 Further Readings

13.0 INTRODUCTION

Gene silencing is the regulation of gene expression in a cell to prevent the expression
of a certain gene. Fundamentally, the ‘Gene Silencing’ can occur either during
transcription or translation, and is often used in research. Quite often the term
gene silencing is considered similar as gene knockdown, because when genes are
silenced, their expression is reduced. In contrast, when genes are knocked out,
they are completely erased from the organism’s genome, and consequently have
no expression. The process of gene silencing aims at inhibiting the expression of
the gene without altering the sequence of the DNA. Gene silencing is possible
both at the transcriptional and translational levels within the cells. Precisely, the
gene silencing involves silencing of promoter sequence in the vicinity of the gene,
preferably caused by hypermethylation of DNA. Genome imprinting, paramutation
and position effect are some of the genetic factors that cause transcriptional gene
silencing. Small non-coding RNA molecules have been reported to mediate
functional Post-Transcriptional Gene Silencing (PTGS) in humans. Recent
investigations in genetics have deciphered the discrete and overlapping sequence
of the proteins involved in the process of non-coding RNA mediated transcriptional
regulation.
Antisense technology is specifically used to manipulate gene expression inside
the cells to treat various diseases. It is considered a powerful tool for studying
gene function utilizing antisense agents to manage the diseases by regulating the
expression of the specific factor that actually causes the particular disease. Highly
specific and effective gene silencing of any disease can be achieved by an accurate
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Gene Silencing and knowledge of the target mRNA sequence and rational design of its complementary
Antisense Technology
antisense agents for the downregulation of its protein message. In particular, methods
used to silence genes are being increasingly used to produce therapeutics to combat
cancer, neurodegenerative disorders and infectious diseases, such as HIV and
NOTES other mutating viral diseases.
In this unit, you will study about the gene silencing and antisense technology
which includes the types and mechanism of gene silencing, genetic factors of gene
silencing, formation of antisense mRNA and inhibition of gene expression by
antisense RNA.

13.1 OBJECTIVES

After going through this unit, you will be able to:

• Understand what gene silencing is
• Explain the mechanism of gene silencing
• Elaborate on genetic factors of gene silencing
• Discuss about the antisense technology
• Analyse the types and formation of antisense mRNA
• Describe inhibition of gene expression by antisense RNA

13.2 GENE SILENCING: AN INTRODUCTION

Gene silencing is the regulation of gene expression in a cell to prevent the expression
of a certain gene. Fundamentally, the ‘Gene Silencing’ can occur either during
transcription or translation, and is often used in research. Quite often the term
gene silencing is considered similar as gene knockdown, because when genes are
silenced, their expression is reduced. In contrast, when genes are knocked out,
they are completely erased from the organism’s genome, and consequently have
no expression. The process of gene silencing aims at inhibiting the expression of
the gene without altering the sequence of the DNA.
Gene silencing is possible both at the transcriptional as well as at the
translational levels within the cells. Typically, the gene silencing involves silencing
of promoter sequence in the vicinity of the gene, preferably caused by
hypermethylation of DNA. Genome imprinting, paramutation and position effect
are some of the genetic factors that cause transcriptional gene silencing. Small
non-coding RNA molecules have been reported to mediate functional Post-
Transcriptional Gene Silencing (PTGS) in humans. Recent investigations in genetics
have deciphered the discrete and overlapping sequence of the proteins involved in
the process of non-coding RNA mediated transcriptional regulation. A considerable
number of mammalian genes have been reported to be regulated by non-coding
RNA at the transcriptional level. Early observations gathered in few decades back
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has revealed non-coding RNA mediated epigenetic changes in tobacco plants. Gene Silencing and
Antisense Technology
The mechanism of TGS is different from that of RNAi (RNA interference) mediated
gene silencing process. TGS induces epigenetic changes which lead to transmission
of the features from the parents to the next generation cells. The process of siRNA
(small or short interfering RNA) mediated TGS is basically a RNA-RNA interaction. NOTES
Unlike in plants, the facts of DNA methylation mediated TGS is not clear for
human genome. Targeting of siRNA or over expression of transgene associated
with encoding siRNA is one of the suitable methods of inducing TGC in cells.
Micro RNAs have been reported to be responsible to provide the endogenous
drive of TGS. Recent reports suggest that a considerable fraction of human genome
encodes for non-coding RNA molecules. Promoter region of the genes preferably
interact with a variety of proteins which initiate the process of TIGS. The siRNA
mediated TGS has been stated to be independent of the process of DNA
methylation. Plant systems utilize RNA Polymerase V transcribed siRNA molecules
which are involved in DNA methylation and TGS. Plant cells undergo TGS
mechanisms which preferably prevent viral infection in the cytoplasm of the cells.
This process is mediated by complementary antisense RNA particles. Genome
imprinting possesses immense importance for the regulation and inactivation of
major disease causing genes in humans. Breeding experiments in mice revealed
that reciprocal translocation heterozygotes carried the mechanism of genome
imprinting. Genome imprinting occurs in the embryo and in the associated tissues
like endosperm and placenta. Alternatively genome imprinting has also been
suggested to have been resulted as an evolutionary effect to prevent transposon
activity in the genome.
The phenomenon of paramutation also induces methylation of DNA or
modification in the structure of ‘Histone’. The allele which induces the change is
known as paramutagenic allele and the allele undergoing the change is called
paramutable allele. The activity of transposons cause structural changes in the
gene sequence thus causing deleterious mutations. However, the phenomenon of
transposon silencing is an essential TGS which prevents the structural disruption
of various important genes. The transgene silencing phenomena imply varied host
defence responses which act to prevent foreign or parasitic elements like
transposable elements, viroids, RNA/DNA viruses or other sources of prokaryotic
DNA. Translocation of the gene into a transcription incompetent heterochromatic
area of the chromatin causes to silence the gene, the position effect. Plant systems
have been exploited for Virus Induced Gene Silencing (VIGS). The main feature
of PTGS is that it decreases the mRNA levels of the homologous genes and
transgenes in the cells. PTGS induces loss of function of the endogenous gene.
One of the major mechanisms of PTGS is associated with non-sense mediated
mRNA degradation where the mRNA (messenger RNA) molecules are degraded
prior to translation. The premature transcript is recognised by a terminal signal
sequence present in them. This method of mRNA degradation involves decapping
of mRNA followed by 5´ to 3´ exonuclease degradation. The microRNAs
(miRNA) are produced from polyadenylated full length precursor pre-miRNA
molecules. The siRNA differ from the miRNA in the fact that the former are more Self-Instructional
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Gene Silencing and specific in binding to particular target mRNAs. The formation of siRNA occurs by
Antisense Technology
cellular machinery similar to that of miRNA. The RISC complex is regulated by
the recognition function of the siRNA which now recognises and cleaves the
complementary mRNA.
NOTES The natural mechanism of gene silencing aims at modulating the temporal
expression of the protein encoding gene. Broadly the process of gene silencing
can be categorized into two types, namely Transcriptional Gene Silencing (TGS)
and Post-Translational Gene Silencing (PTGS). PTGS is the product of transcribed
mRNA of a specific gene being silenced. When mRNA was destructed, then
translation to form an active gene product (in most cases, a protein) will be
prevented. A general process of PTGS silencing is by RNAi.
13.2.1 Transcriptional Gene Silencing (TGS)
This category of gene silencing involves silencing of promoter sequence in the
vicinity of the gene, preferably caused by hypermethylation of DNA. Due to this
the promoter sequence is inaccessible to the transcription initiating factors.
Therefore, the ‘Histone’ modification appears as a key mechanism for regulating
Transcriptional Gene Silencing (TGS). Typically, the factors namely the genome
imprinting, paramutation and position effect are considered as some of the significant
genetic factors that cause TGS. Small non-coding RNA molecules have been
reported to mediate functional Post-Transcriptional Gene Silencing (PTGS) in
humans. However, TGS is different from RNAi silencing in terms of its long term
stability and epigenetic modifications. The epigenetic modifications are transmitted
from the parents to daughter cells. Recent investigations in genetics have deciphered
the discrete and overlapping sequence of the proteins involved in the process of
non-coding RNA mediated transcriptional regulation. A considerable number of
mammalian genes have been reported to be regulated by non-coding RNA at the
transcriptional level. Early observations gathered in few decades back has revealed
non-coding RNA mediated epigenetic changes in Tobacco plants. Arabidopsis
has also been reported to exhibit the mechanism of TGS. The phenomenon of
non-coding RNA mediated TGS has been investigated in a variety of model
organisms, such as Arabidopsis thaliana, Saccharomyces pombe, Drosophila
melanogaster and Caenorhabditis elegans. Nuclear run on analysis has revealed
that exogenous siRNA has been effective in creating TGS.
Mechanism of Non-Coding RNA Mediated TGS
The mechanism of TGS is different from that of RNAi mediated gene silencing
process. TGS induces epigenetic changes which lead to transmission of the features
from the parents to the next generation cells. Recent findings have led to the
conclusion that two separate groups of siRNAs are capable of operating at the
promoter region and exon region of the coding transcript. This leads to the
functionality of TGS in the cells. The promoter directed siRNA interact with a low
level expressed promoter associated RNA which is present at the 5’ UTR region
of the gene. Thus the process of siRNA mediated TGS is basically a RNA-RNA
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interaction. Unlike in plants, the facts of DNA methylation mediated TGS is not Gene Silencing and
Antisense Technology
clear for human genome. Targeting of siRNA or over expression of transgene
associated with encoding siRNA is one of the suitable method of inducing TGS in
cells. The microRNAs or miRNAs have been reported to be responsible to provide
the endogenous drive of TGS. Recent reports suggest that a considerable fraction NOTES
of human genome encodes for non-coding RNA molecules. These RNA species
appear to function as antisense fragments for several coding mRNA. Cell culture
studies have been implied to clarify the mechanisms of siRNA mediated TGS in
human cells. Promoter region of the genes preferably interact with a variety of
proteins which initiate the process of TGS. Basically, the siRNA mediated TGS
has been stated to be independent of the process of DNA methylation. Plant systems
utilize RNA Polymerase-(V) transcribed siRNA molecules which are involved in
DNA methylation and TGS. Plant cells undergo TGS mechanisms which preferably
prevent viral infection in the cytoplasm of the cells. This process is mediated by
complementary antisense RNA particles.
13.2.2 Genetic Factors Associated with Transcriptional Gene Silencing
(TGS)
The following genetic factors are responsible for the Transcriptional Gene Silencing
(TGS).
Genome Imprinting
This mechanism of gene silencing is an epigenetic phenomenon which operates in a
parent-of-origin specific mode of transmission. Plants, animals and fungi have been
reported to exhibit the phenomenon of genome imprinting. This epigenetic
phenomenon is an extra Mendelian Mode of Inheritance which is
characteristically regulated by the DNA and the Histone Methylation. More
than hundred genes have been reported to be imprinted, for example in mouse
genome. The epigenetic changes are imprinted in the germline cells of the parents
and fixed in the next generation somatic cells which proliferate through mitotic cell
division.
Genome imprinting possesses immense importance for the regulation and
inactivation of major disease causing genes in humans. Breeding experiments in
mice revealed that reciprocal translocation heterozygotes carried the mechanism
of genome imprinting. Induced imprinting has led to development of parthenogenetic
mouse which contain two sets of maternal chromosomes. The paternal set of genes
were imprinted or silenced. The evolutionary significance of the imprinted genes
has been discussed by geneticists. They refer it to be result of antagonistic coevolution
guided by positive selection pressure. Genome imprinting is largely regulated by
the environmentally induced factors like atmospheric pollutants, diet and life style
of humans. The phenomenon of genome imprinting has independently evolved in
mammals and flowering plants. Genome imprinting occurs in the embryo and
associated tissues like endosperm and placenta. Alternatively, the genome imprinting
has also been suggested to have been resulted as an evolutionary effect to prevent
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Gene Silencing and transposon activity in the genome. The differential imprinting of Maternal and
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Paternal Genes regulates the magnitude of nutrient flow within the plant embryo.
The concept of parental conflict appears from the differential imprinting of the
maternal and paternal genes. Figure 13.1 illustrates the mechanism of gene silencing
NOTES by the Histone, the DNA Methylation and the miRNA applications.

Fig. 13.1 Mechanism of Gene Silencing by Histone,

DNA Methylation and miRNA Applications

Paramutation
In epigenetics, a paramutation is defined as an interaction between two alleles at a
single locus, whereby one allele induces a heritable change in the other allele. As
per the standard definition, “In the paramutation mechanism, the change may be in
the pattern of DNA Methylation or Histone modifications. The allele inducing the
change is termed as ‘paramutagenic’, while the allele that has been epigenetically
altered is termed as ‘paramutable’.”
Principally, the phenomenon of allelic interaction is within a single locus
which results in the heritable change in a particular allele of the locus. The
phenomenon of paramutation also induces methylation of DNA or modification in
the structure of Histone. The allele which induces the change is known as
‘paramutagenic allele’ and the allele undergoing the change is called ‘paramutable
allele’.
A paramutable allele may have altered levels of gene expression, which
may continue in offspring which inherit that allele, even though the paramutagenic
allele may no longer be present. Through proper breeding, paramutation can result
in sibling plants that have the same genetic sequence, but with drastically different
phenotypes.
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 Characteristically, the paramutagenic allele induces an altered level of gene Gene Silencing and
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expression in the organism which exhibit difference in the phenotypes. Typically,
the gene sequence remains similar in the wild type and paramutagenic genes. The
preliminary report of the phenomenon of paramutation was investigated in Maize
kernels. Maize kernels have been reported to weakly express the ‘Red 1 Locus’. NOTES
Recent findings reveal that paramutagenic interaction may occur between the two
homologous transgenes.
The phenomenon of paramutation has also been observed in Drosophila
melanogaster and mice. Animal and fungal systems have been observed to exhibit
meiotically heritable traits caused due to paramutation. Thus, paramutation is an
important non-Mendelian trait in organisms responsible for causing TGS and
subsequent change in phenotype. The paramutation phenomenon in Maize Loci,
such as the Pl1, p1, r1, and b1 are associated with Anthocyanin Biosynthesis. The
genes encode for transcription factors necessary to induce anthocyanin biosynthesis.
In the case of paramutation, the affected paramutable allele is highly expressed. In
heterozygous combination one allele is completely silenced due to paramutation.
The silenced allele therefore does not produce functional transcript necessary for
protein expression. A cross between the plants containing paramutagenic allele
with that having paramutable allele may result in the silencing of an allele in the filial
generation.
Transposon Silencing
The activity of transposons cause structural changes in the gene sequence thus
causing deleterious mutations. However, the phenomenon of transposon silencing
is an essential TGS which prevents the structural disruption of various important
genes. The activity of transposons results in the occurrence of various autoimmune
diseases. The activity of siRNA has been deciphered to be responsible for
transposon silencing. The germline tissues preferably contain the prevalence of
siRNA activity which silence the expression of transposons in the next generation.
The Piwi-interacting RNA (piRNA) are the largest class of siRNA of about 26-31
nucleotide long which interact with the Piwi family proteins. Various Piwi-RNA
have been reported to be associated with the centromeric and telomeric region of
the chromosome. These piRNAs are antisense to the mRNA encoded by
transposon elements, thus causing silencing of the genes. The phenomenon of
transposon silencing has been observed in Drosophila melanogaster, mice and
Arabidopsis species.
Fundamentally, the Piwi or PIWI genes were identified as regulatory proteins
responsible for stem cell and germ cell differentiation. PIWI is an abbreviation of
P-element Induced WImpy testis in Drosophila melanogaster. Piwi proteins are
highly conserved RNA-binding proteins and are present in both plants and animals.
Transgene Silencing
The transgene silencing phenomena imply varied host defence responses which act
to prevent foreign or parasitic elements like transposable elements, viroids, RNA/
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Gene Silencing and DNA viruses or other sources of prokaryotic DNA. Transgenes inserted into a
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cell also trigger similar responses. The host defense response manifested by
transgene silencing may act at the genome level and induce DNA methylation.
Another mechanism, of transgene silencing operates at the post-transcriptionally
NOTES level. Thus TGS may involve transgene silencing as an important mode of host
defense. The process of transgene silencing involves DNA-RNA interaction. The
transgene silencing method is one of the widely used siRNA strategy to inhibit
gene expression at the TGS level. This process finds application in modulating
gene expression associated to developmental phases, pathogen attack or abiotic
stress. Rearrangement or duplication of transgenes incorporated within the host
genome possesses changes of getting silenced. Enhancing the effect of transgene
by doubling its dose in the homozygous transgenic lines result in higher chances of
gene silencing. The transgene DNA can be delivered by means of a bacterial or
viral vector entered within the host cell. The Retrovirus Vectors are prepared by
replacing the viral genes by replacing it with the transgene of interest. The
recombinant genome is then packed into the virus particle. In humans, this process
is a prevalent mode of stem cell therapy and gene silencing.
Position Effect
The expression of a gene can be altered due to the act of translocation within
chromosomal segments. The eye colour trait in Drosophila melanogaster is a
well-documented example of position effect. The position effect in this case is
manifested by the variegated red eye phenotype. The deleterious outcome of
position effect has been associated within the occurrence of certain diseases in
humans. The euchromatin and heterochromatin part of the chromosome are
cytologically distinguishable and they differ in their transcriptional ability. The
alteration in the position of the gene in the genome leads to variation in the gene
environment. The transcriptional activity of the gene mostly depends upon the
functioning of the promoter and the enhancer. The absence of the enhancer in the
altered position of the gene results in its silencing. Alternately, the presence of an
enhancer can silence the gene and cause its unusual expression within a tissue. The
variegation effect may result from the gene being translocate in the vicinity of another
gene with the common regulatory element which governs the expression of both
the genes. Translocation of the gene into a transcription incompetent
heterochromatic area of the chromatin causes to silence the gene. The variegation
effect exhibited by the PAX6 gene provide concrete evidence of position effect
associated with human disease. The translocation of this gene leads to haploid
insufficiency and deletion which results in point mutation. Thus, the position effect
of the gene leads to formation of ‘Aniridia’ - a rare, sight-threatening disorder that
affects the cornea, iris, intraocular pressure, lens, fovea, and optic nerve. Figure
13.2 illustrates the mechanism of position effect in the model organism Drosophila
melanogaster.
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NOTES

Fig. 13.2 Mechanism of Position Effect in Drosophila melanogaster

RNA-Directed DNA Methylation

This epigenetic phenomenon was discovered in plants where the dsRNA (double-
strand RNA) molecules are processed to produce 21-25 nucleotide long siRNAs.
These siRNA molecules induce DNA methylation thus resulting in gene silencing.
Arabidopsis has been reported to exhibit this epigenetic phenomenon. Plants
systems involve various modes of generation of siRNA which involve viral replication
intermediate nucleotide fragments, products formed by endogenous RNA directed
RNA polymerase, transcribed inverted repeat fragments and transposable elements.
Apart from RNA molecules various proteins, namely Agronaute, DNA
Methyltransferases and plant specific Polymerases (IV and V) are responsible for
RNA directed DNA Methylation. Cytosine in plant DNA fragments are more
susceptible to de novo methylation. The RNA viroids are one of the important
regulators of RNA-directed DNA methylation in plants. Typically, the RNA viroids
or viruses produce only RNA particles during the replicating phase of their life
cycle. Experimental analysis of transgenic plants producing higher amount of dsRNA
have confirmed that dsRNA itself regulate RNAi and DNA methylation in plant
genomes. In plant and animal genomes, the cytosine regions are preferably
methylated by the dsRNA molecules. Cytosine Methyltransferase is a major
molecule regulating Cytosine Methylation. The dsRNA introduced in the cells are
successful in inducing RNAi in Caenorhabditis elegans. Amazingly, the fission
yeasts have been reported to exhibit RNA-directed Heterochromatin formation
in their ‘Genome’. Cytosine Methylation mechanism is lacking in this organism
and alternately Histone molecules regulate the process of Heterochromatin
formation. Similar observations have been observed in the case of Drosophila
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Gene Silencing and melanogaster. Figure 13.3 illustrates the mechanism of dsRNA formation for
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TGS.

NOTES

Fig. 13.3 Mechanism of dsRNA Formation for TGS

13.2.3 Post-Transcriptional Gene Silencing (PTGS)

The introduction of transgenes or double stranded RNA molecules lead to induction
of Post-Transcriptional Gene Silencing (PTGS) in the homologous genes or
transgenes present in the genome of the organism. PTGS induced by the
introduction of transgene has been reported on plants (co-suppression) and also
in Fungi (quelling). RNA interference (RNAi) induced by dsRNA has been reported
in animal system. Plant systems have been exploited for Virus Induced Gene
Silencing (VIGS). The main feature of PTGS is that it decreases the mRNA levels
of the homologous genes and transgenes in the cells. PTGS induces loss of function
of the endogenous gene. Apart from plant systems, Neurospora (a genus of
Ascomycete Fungi) or Caenorhabditis elegans have also been reported to
successfully exhibit the phenomenon of PTGS induced by transgene application.
The phenomenon of PTGS does not inhibit transcription but degrade or silence
the effective mRNA molecules. This can be correlated with increased formation
of degraded of RNA intermediates arising as a result of mRNA silencing. One of
the major mechanisms of PTGS is associated with non-sense mediated mRNA
degradation where the mRNA molecules are degraded prior to translation. The
premature transcript is recognised by a terminal signal sequence present in them.
This method of mRNA degradation involves decapping of mRNA followed by 52
to 32 exonuclease degradation. The small RNA molecules in plants have been
reported to be up to 25 nucleotide in length and capable of inducing gene silencing.
The gene Dicer has been reported to be responsible for producing short fragment
of dsRNA necessary for RNAi in Drosophila melanogaster. The Dicer gene
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encodes for the RNAse III type enzyme which cleaves the dsRNA into short Gene Silencing and
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fragments of siRNA (21-25 nucleotide long).
Components and Mechanism of RNA Interference
In the RNA Interference or RNAi, the type of PTGS is induced by the application NOTES
of dsRNA which produces siRNA (small interfering RNA) necessary for silencing
of the homologous genes. The preliminary evidence of RNAi was observed from
investigations in Caenorhabditis elegans which was later also evident in
Drosophila melanogaster. The method was earlier termed as co-suppression or
quelling. The RNAi method involves the function of two species of RNA termed
as miRNA (microRNA) and siRNA. The phenomenon of RNAi is an important
defence mechanism against the entry of parasitic nucleotide fragments in plants
which are mainly virus and transposons.
miRNA: The microRNAs (miRNA) are produced from polyadenylated full length
precursor pre-miRNA molecules. The precursor of miRNA is the ssRNA (single-
stranded RNA) which preferably produces a hairpin like secondary structure.
The miRNA may not exhibit complete complementarities to the functional mRNA
fragments. Additionally, the miRNA molecules are capable of inducing gene silencing
mainly by triggering Heterochromatin formation in the ‘Genome’. The dsRNA
precursor of miRNA is cleaved through Dicer to produce mature miRNA which is
incorporated into the RISC complex (RNA-Induced Silencing Complex). Certain
3´UTR regions of mRNA molecules possesses certain regulatory domains which
function for RNA interference. The 3´UTR regions contain binding sites for miRNA
and certain regulatory proteins. The binding of miRNA to this regulatory region
triggers inhibition of translation in various mRNA molecules.
siRNA: The small interfering RNAs (siRNA) are important component of PTGS
mediated by RNA interference. The siRNA are 21-25 nu. In length and sensitive
or specific to the target mRNA for its binding. The high sensitivity of siRNA,
however, may be hampered by a single nucleotide mismatch between the mRNA
and siRNA. The structure of siRNA contains a 5´ Phosphate group, 3´ Hydroxyl
group and a 2-3 Nucleotide long 3´ overhang. The siRNA differ from the miRNA
in the fact that the former are more specific in binding to particular target mRNAs.
The formation of siRNA occurs by cellular machinery similar to that of miRNA.
Dicer cleaves the dsRNA to produce the fragments of siRNA. The siRNA once
formed gets associated with other proteins to form the RISC complex where it
associates with miRNA. The RISC complex is regulated by the recognition function
of the siRNA which now recognises and cleaves the complementary mRNA.
Transfection or electroporation method can be successfully applied to introduce
siRNA within the cells of plants or animals. The process of high accuracy delivery
is mediated by the cationic liposomes which pass across the membrane. Adenovirus
or Retrovirus mediated recombinant viral vectors can also be implied to deliver
siRNA into the cells.

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Gene Silencing and Dicer: These are dsRNA specific RNAase Type III Ribonucleases which function
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to cleave the precursor RNA molecules to produce miRNA or siRNA. The Dicer
family proteins are essentiallyATP-dependent nucleases. Typically, the Dicer family
proteins are essentially found in various organisms like yeast, Drosophila
NOTES melanogaster, human, Arabidopsis thaliana and fungal members. The structure
of this ribonuclease consists of dual RNAse III Motif in the Carboxyl terminal and
a dsRNA binding domain. An additional PAZ domain is necessary for undergoing
protein interaction with Agronaute of Piwi RNA.
RNA-Inducing Silencing Complex (RISC): RISC is a large sized 500 kDa
multi-protein complex which functions to cleave mRNA which is recognized by
siRNA. The miRNA and siRNA are the main regulatory components of RISC.
The complex of RISC involves the unwinding of the ds-siRNA complex by Helicase.
The endonuclease component of RISC is Agronaute protein which cleaves the
mRNA sequences bound to the siRNA.

13.3 FORMATION OF ANTISENSE mRNA AND

INHIBITION OF GENE EXPRESSION

Among various types of RNA interference inducing molecules namely formation

of antisense mRNA, inhibition of gene expression by antisense RNA, formation of
siRNA and miRNA are important for inhibition of gene expression. Antisense
RNA are unique DNA transcripts of 20-25 nucleotide long that complement the
mRNA in the cell. The advent of antisense RNA technology shall gradually replace
the method of traditional gene-specific silencing method. Investigations on miRNAs,
siRNAs, lncRNAs (long non-coding RNAs), and piRNAs (Piwi-interacting) have
deciphered the process of their formation and regulatory action which modulate
the phenomenon of gene silencing.
Formation and Regulation of miRNA
1. The miRNAs were initially identified in the model system Caenorhabditis
elegans. The miRNA is the ssRNA of 18-25 nu long transcript different
from the other long RNA transcripts of non-coding nature.
2. The precursor of miRNA is termed as pre-miRNA. The miRNA derived
from this precursor RNA cannot be transcribed and is used as an inhibitor
of its corresponding coding gene.
3. The precursor RNA is processed in the nucleus by the activity of RNase III
(i.e., Drosha and DGCR8/Pasha) to generate pre-miRNA.
4. The intra-nuclear pre-miRNA is transferred to the cytoplasm by the action
of Exportin-5.
5. The pre-miRNA is transferred to the cytoplasm and followed by this step it
forms a secondary structure with stem and loop.
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 6. The pre-miRNA is cleaved at the hairpin structure by Dicer (RNAase) to Gene Silencing and
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generate miRNAs.
7. The miRNAs thus formed associates with the Agronaute family of proteins
of the RISC complex and functions for silencing mRNA molecules. A single
NOTES
mRNA can be silenced by multiple numbers of miRNA molecules. The
pairing of the mRNA-miRNA strands occurs only for a region of 20
nucleotides.
8. Thus miRNA are capable of functioning to silence a diverse number of
coding genes.
Formation and Regulation of siRNA
1. The siRNA are dsRNA of 20 nu long which are synthesized in vivo and
transported from the nucleus to the cytoplasm by means of transporters.
2. Artificially prepared siRNA can also be applied in vitro into the cells.
3. The dsRNA is transported from the nucleus mediated by transmembrane
protein SID-1.
4. The transported form of siRNA is edited by DCR-1 to form ds-siRNA.
5. The ds-siRNA after their formation remain associated with RISC complex.
6. The RDE-2 and MUT-7 proteins facilitate the siRISC to associate with the
coding mRNA thus causing its silencing.
7. Antisense siRNA are also used as primers in the process of RNA-dependent
RNA polymerase reaction. This reaction uses mRNA as a template for
synthesizing siRNA.
8. The dsRNAs can also be obtained from virus or transposon activation or
specific repetitive sequences.
9. The miRNA is partially complementary to mRNA in the 3’UTR region.
However, siRNA is completely complementary to the mRNA.
Formation and Regulation of lncRNAs
1. This type of RNA ranges from 200 bp - 1000 kb in length. These molecules
are sense, antisense, bidirectional or intronic in nature.
2. The lncRNAs are transcribed by RNA Polymerase (II) which undergoes
post-transcriptional modifications like 5'-capping, Polyadenylation and pre-
lncRNA splicing.
3. The lncRNAs fold to form secondary or tertiary structures. Moreover, the
lncRNAs combine with the gene transcripts to form dsRNA.
4. The double stranded complex is cleaved by Dicer to produce endogenous
siRNAs.
5. Antisense lncRNAs function in X-Chromosome inactivation and genome
imprinting.
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Gene Silencing and Formation and Regulation of piRNAs
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1. piRNA are 26-32 nu long fragments which differ from the siRNA and miRNA
in terms of their function, expression and structure.
NOTES 2. The piRNA in association with the PIWI protein involve in gene silencing.
3. Long sized ssRNA transcripts are presumed to be precursors for piRNA.
4. The endonucleases process the long fragments of ssRNA to form 5´ end of
the mature piRNA.
5. The piRNA have also been suggested to be formed from double stranded
DNA-RNA strand which is modified by reverse transcriptase of retro-
transposons.
6. The piRNA regulate transcription, mRNA stability and gene translation.
Figure 13.4 illustrates the mechanism of miRNA and siRNA formation and RISC
mediated RNAi pathway.

Fig. 13.4 Mechanism of miRNA and siRNA Formation and

RISC Mediated RNAi Pathway
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Role of Antisense mRNA in Gene Silencing Gene Silencing and
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The antisense RNA molecules are implied in prevention of various human diseases.
The parasitic nucleotides sequences are defended by the cellular antisense RNA
which prevent the expression of the foreign gene segments. Apart from human NOTES
diseases antisense technology is an important tool for plant biotechnology.
Manipulation of biosynthetic pathways, regulation of male sterile lines and post
harvest management of fruits are some of the important instances of gene silencing
mediated by antisense technology.
Effect in Antivirus Treatment
Human Immunodeficiency Various (HIV), Hepatitis C Virus (HCV) and Influenza
Virus (IV) infection has been reported to be modulated by antisense technology.
HIV-1 replication has been reported to be inhibited by a recombinant Lentiviral
Vector which has been constructed based on HIV 1. The recombinant Lentivirus
Vector contains 937 bp long antisense sequence which represses the transcripts
of HIV1 envelop protein. Antisense RNA has also been implied to inhibit translation
of HCV RNAs within the human Hepatocellular Carcinoma Cells. The antisense
RNA have been prepared on the complementarity of 5' region of the HCV genome
between the Genetic Loci 1–402. The siRNA expression plasmid pBabe-Super
has been used to inhibit the infection of influenza virus in chicken eggs and BALB/
c mice. Thus, antisense RNA method appears to be a therapy against virus infection
in humans and animals.
Effect on Anticancer Treatment
A substantiating amount of information has been obtained on the role of antisense
RNA in regulating the growth and proliferation of cancerous or malignant cell
lines. The ncRNAs (non-coding RNA) serve as the potential biomarkers for cancer.
The expression profile for various miRNAs is indicators of the status of cancerous
cells. Antisense RNA has been implied to inhibit the Stat5 or Survivin expression
in cancerous tumours. This results in the apoptosis of tumours and termination of
malignancy. Antisense RNA has been reported to be associated with the regulation
of cancer-promoting genes like miR-21, miR-155, miR-17-20 and cancer-
suppressing genes like miR-15, miR-16 and miR-143.
Regulation of Ethylene Biosynthesis and Post-Harvest Fruit Management
The biosynthetic pathway of Ethylene has been reported to be modulated by
antisense technology in climacteric fruits. The surge in Ethylene Biosynthesis in
ripening fruits results in increased activity of cell wall loosing enzymes and sugar
metabolism. Thus, repression of Ethylene Accumulation by Antisense RNA of 1-
AminocyClopropane-1-Carboxylate (ACC) Oxidase delays fruit ripening.
Antisense technology thus helps to manage post harvest storage of fruits and
increase shelf life. The ‘Flavr Savr Tomato’ has been obtained as a modified
transgenic tomato with repressed activity of Pectin Methyl Esterase which prevents
or delays cell wall loosening, thus delaying ripening.
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Gene Silencing and Treatment of Viral Infection in Plants
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Antisense RNA technology has been applied in plants to inhibit the process of
viral RNA biosynthesis. Antisense RNA technology has been applied in various
NOTES model crop plants like Tomato, Tobacco and Maize, where the viral infection has
been successfully inhibited. Antisense RNA has been implied for repressing the
expression of DNA of the AL1-Coding Gene from the geminivirus Tomato Golden
Mosaic Virus (TGMV). Similar efforts have been able to regulate the infection of
Tobacco Mosaic Virus (TMV) and Cauliflower Mosaic Virus (CaMV).
Applications in Crop Management
Amylose free diet Potato, coloured Petunia and management of male sterility are
some of the major instances of antisense RNA technology. Male sterility can be
implied for successful production of hybrids by preventing the act of self-pollination,
also termed as selfing. Antisense technology is used for restoring male fertility or
inducing male sterility in the crops desired to be hybridized. The application of
antisense Chalcone Synthase (CHS) gene inhibits flavonoid biosynthesis and
induces male sterility in Petunia varieties. Silencing of the Mutator S (MutS)
homolog in Tomato has also led to induction of cytoplasmic sterility.
Regulation of Microbial Metabolism
Antisense RNA technology faces limitations in its applications in microbial
metabolism. The genetic complexity of microbes render a challenge in the success
of antisense RNA in microbial gene silencing. Increased Thaumatin production
has been achieved by expression of antisense strand of pepB encoding
aspergillopepsin B from Aspergillus awamori. Additionally, the Xylitol
production in Trichoderma (is a genus of fungi in the family Hypocreaceae) has
been achieved by the antisense RNA production to silent the expression of Xylitol
Dehydrogenase 1 (xdh1). Antisense RNA mediated attenuation of pga1 encoding
heterotrimeric G protein α-subunit in Penicillium chrysogenum has revealed
its important role in cell growth and colony formation.

Check Your Progress

1. Explain gene silencing.
2. What are the categories of the gene silencing?
3. What does Transcriptional Gene Silencing (TGS) category of gene silencing
involve?
4. Give examples of non-coding RNA for TGS.
5. Define paramutation.
6. What is Post-Transcriptional Gene Silencing (PTGS)?

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 Gene Silencing and
13.4 ANSWERS TO CHECK YOUR PROGRESS Antisense Technology

QUESTIONS

1. Gene silencing is the regulation of gene expression in a cell to prevent the NOTES
expression of a certain gene. Fundamentally, the ‘Gene Silencing’ can occur
either during transcription or translation, and is often used in research. The
process of gene silencing aims at inhibiting the expression of the gene without
altering the sequence of the DNA.
Gene silencing is possible both at the transcriptional as well as at the
translational levels within the cells. Typically, the gene silencing involves
silencing of promoter sequence in the vicinity of the gene, preferably caused
by hypermethylation of DNA. Genome imprinting, paramutation and position
effect are some of the genetic factors that cause transcriptional gene silencing.
The natural mechanism of gene silencing aims at modulating the temporal
expression of the protein encoding gene.
2. Broadly the process of gene silencing can be categorized into two types,
namely Transcriptional Gene Silencing (TGS) and Post-Translational Gene
Silencing (PTGS). PTGS is the product of transcribed mRNA of a specific
gene being silenced.
3. Transcriptional Gene Silencing (TGS) category of gene silencing involves
silencing of promoter sequence in the vicinity of the gene, preferably caused
by hypermethylation of DNA. Due to this the promoter sequence is
inaccessible to the transcription initiating factors. Therefore, the ‘Histone’
modification appears as a key mechanism for regulating Transcriptional Gene
Silencing (TGS). Typically, the factors namely the genome imprinting,
paramutation and position effect are considered as some of the significant
genetic factors that cause TGS.
4. Early observations gathered in few decades back has revealed non-coding
RNA mediated epigenetic changes in Tobacco plants. Arabidopsis has also
been reported to exhibit the mechanism of TGS. The phenomenon of non-
coding RNA mediated TGS has been investigated in a variety of model
organisms, such as Arabidopsis thaliana, Saccharomyces pombe,
Drosophila melanogaster and Caenorhabditis elegans. Nuclear run on
analysis has revealed that exogenous siRNA has been effective in creating
TGS.
5. The paramutation is defined as an interaction between two alleles at a single
locus, whereby one allele induces a heritable change in the other allele. As
per the standard definition, “In the paramutation mechanism, the change
may be in the pattern of DNA Methylation or Histone modifications. The
allele inducing the change is termed as ‘paramutagenic’, while the allele that
has been epigenetically altered is termed as ‘paramutable’.”
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Gene Silencing and 6. The introduction of transgenes or double stranded RNA molecules lead to
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induction of Post-Transcriptional Gene Silencing (PTGS) in the homologous
genes or transgenes present in the genome of the organism. PTGS induced
by the introduction of transgene has been reported on plants (co-
NOTES suppression) and also in Fungi (quelling). RNA interference (RNAi) induced
by dsRNA has been reported in animal system. Plant systems have been
exploited for Virus Induced Gene Silencing (VIGS). The main feature of
PTGS is that it decreases the mRNA levels of the homologous genes and
transgenes in the cells. PTGS induces loss of function of the endogenous
gene. The phenomenon of PTGS does not inhibit transcription but degrade
or silence the effective mRNA molecules.

13.5 SUMMARY

• Gene silencing is the regulation of gene expression in a cell to prevent the

expression of a certain gene. Fundamentally, the ‘Gene Silencing’ can occur
either during transcription or translation, and is often used in research.
• Quite often the term gene silencing is considered similar as gene knockdown,
because when genes are silenced, their expression is reduced. In contrast,
when genes are knocked out, they are completely erased from the organism’s
genome, and consequently have no expression.
• The process of gene silencing aims at inhibiting the expression of the gene
without altering the sequence of the DNA.
• Gene silencing is possible both at the transcriptional as well as at the
translational levels within the cells. Typically, the gene silencing involves
silencing of promoter sequence in the vicinity of the gene, preferably caused
by hypermethylation of DNA.
• Genome imprinting, paramutation and position effect are some of the genetic
factors that cause transcriptional gene silencing.
• The natural mechanism of gene silencing aims at modulating the temporal
expression of the protein encoding gene.
• Broadly the process of gene silencing can be categorized into two types,
namely Transcriptional Gene Silencing (TGS) and Post-Translational Gene
Silencing (PTGS). PTGS is the product of transcribed mRNA of a specific
gene being silenced.
• Transcriptional Gene Silencing (TGS) category of gene silencing involves
silencing of promoter sequence in the vicinity of the gene, preferably caused
by hypermethylation of DNA. Due to this the promoter sequence is
inaccessible to the transcription initiating factors. Therefore, the ‘Histone’
modification appears as a key mechanism for regulating Transcriptional Gene
Silencing (TGS).

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• Typically, the factors namely the genome imprinting, paramutation and position Gene Silencing and
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effect are considered as some of the significant genetic factors that cause
TGS.
• Small non-coding RNA molecules have been reported to mediate functional
NOTES
Post-Transcriptional Gene Silencing (PTGS) in humans. However, TGS is
different from RNAi silencing in terms of its long term stability and epigenetic
modifications.
• The phenomenon of non-coding RNA mediated TGS has been investigated
in a variety of model organisms, such as Arabidopsis thaliana,
Saccharomyces pombe, Drosophila melanogaster and Caenorhabditis
elegans.
• The mechanism of TGS is different from that of RNAi mediated gene silencing
process. TGS induces epigenetic changes which lead to transmission of the
features from the parents to the next generation cells.
• The microRNAs or miRNAs have been reported to be responsible to provide
the endogenous drive of TGS.
• Promoter region of the genes preferably interact with a variety of proteins
which initiate the process of TGS. Basically, the siRNA mediated TGS has
been stated to be independent of the process of DNA methylation.
• Plant systems utilize RNA Polymerase-(V) transcribed siRNA molecules
which are involved in DNA methylation and TGS.
• Genome imprinting mechanism of gene silencing is an epigenetic phenomenon
which operates in a parent-of-origin specific mode of transmission.
• Plants, animals and fungi have been reported to exhibit the phenomenon of
genome imprinting. This epigenetic phenomenon is an extra Mendelian Mode
of Inheritance which is characteristically regulated by the DNA and the
Histone Methylation.
• Genome imprinting occurs in the embryo and associated tissues like
endosperm and placenta. Alternatively, the genome imprinting has also been
suggested to have been resulted as an evolutionary effect to prevent
transposon activity in the genome.
• The differential imprinting of Maternal and Paternal Genes regulates the
magnitude of nutrient flow within the plant embryo.
• The paramutation is defined as an interaction between two alleles at a single
locus, whereby one allele induces a heritable change in the other allele.
• As per the standard definition, “In the paramutation mechanism, the change
may be in the pattern of DNA Methylation or Histone modifications. The
allele inducing the change is termed as ‘paramutagenic’, while the allele that
has been epigenetically altered is termed as ‘paramutable’.”
• A cross between the plants containing paramutagenic allele with that having
paramutable allele may result in the silencing of an allele in the filial generation.
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Gene Silencing and • The phenomenon of transposon silencing is an essential TGS which prevents
Antisense Technology
the structural disruption of various important genes. The activity of
transposons results in the occurrence of various autoimmune diseases.
• The Piwi-interacting RNA (piRNA) are the largest class of siRNA of about
NOTES
26-31 nucleotide long which interact with the Piwi family proteins. Various
Piwi-RNA have been reported to be associated with the centromeric and
telomeric region of the chromosome.
• The transgene silencing phenomena imply varied host defence responses
which act to prevent foreign or parasitic elements like transposable elements,
viroids, RNA/DNA viruses or other sources of prokaryotic DNA.
• The host defense response manifested by transgene silencing may act at the
genome level and induce DNA methylation. Another mechanism, of
transgene silencing operates at the post-transcriptionally level.
• The process of transgene silencing involves DNA-RNA interaction. The
transgene silencing method is one of the widely used siRNA strategy to
inhibit gene expression at the TGS level.
• RNA-directed DNA Methylation is an epigenetic phenomenon. It was
discovered in plants where the dsRNA (double-strand RNA) molecules
are processed to produce 21-25 nucleotide long siRNAs. These siRNA
molecules induce DNA methylation thus resulting in gene silencing.
Arabidopsis has been reported to exhibit this epigenetic phenomenon.
• The introduction of transgenes or double stranded RNA molecules lead to
induction of Post-Transcriptional Gene Silencing (PTGS) in the homologous
genes or transgenes present in the genome of the organism.
• PTGS induced by the introduction of transgene has been reported on plants
(co-suppression) and also in Fungi (quelling).
• The main feature of PTGS is that it decreases the mRNA levels of the
homologous genes and transgenes in the cells. PTGS induces loss of function
of the endogenous gene. The phenomenon of PTGS does not inhibit
transcription but degrade or silence the effective mRNA molecules.
• The microRNAs (miRNA) are produced from polyadenylated full length
precursor pre-miRNA molecules.
• The small interfering RNAs (siRNA) are important component of PTGS
mediated by RNA interference.
• Antisense RNA are unique DNA transcripts of 20-25 nucleotide long that
complement the mRNA in the cell.
• The advent of antisense RNA technology shall gradually replace the method
of traditional gene-specific silencing method. Investigations on miRNAs,
siRNAs, lncRNAs (long non-coding RNAs), and piRNAs (Piwi-interacting)
have deciphered the process of their formation and regulatory action which
modulate the phenomenon of gene silencing.
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 Gene Silencing and
13.6 KEY WORDS Antisense Technology

• Gene silencing: It refers to the regulation of gene expression in a cell to

prevent the expression of a certain gene, it can occur either during NOTES
transcription or translation.
• Transcriptional Gene Silencing (TGS): This category of gene silencing
involves silencing of promoter sequence in the vicinity of the gene, preferably
caused by hypermethylation of DNA.
• Genome imprinting: This mechanism of gene silencing is an epigenetic
phenomenon which operates in a parent-of-origin specific mode of
transmission.
• Paramutation: It is the interaction between two alleles at a single locus,
whereby one allele induces a heritable change in the other allele.
• miRNA: The microRNAs (miRNA) are produced from polyadenylated
full length precursor pre-miRNA molecules.
• siRNA: The small interfering RNAs (siRNA) are important component of
PTGS mediated by RNA interference.

13.7 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is gene silencing? What are its types?
2. Explain the term Transcriptional Gene Silencing (TGS).
3. Define the terms genome imprinting, paramutation and transposon silencing.
4. What is Piwi-interacting RNA (piRNA)?
5. Define the term PTGS.
6. What is Dicer?
7. What is antisense mRNA?
8. Explain regulation process of microbial metabolism.
Long Answer Questions
1. Briefly discuss about the gene silencing methodology giving its significance
and mechanism.
2. Discuss the Transcriptional Gene Silencing (TGS) with reference to the
mechanism of non-coding RNA mediated TGS giving appropriate examples.
3. Explain briefly the genetic factors that are associated with Transcriptional
Gene Silencing (TGS).
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Gene Silencing and 4. Discuss the mechanism of RNA-directed DNA Methylation.
Antisense Technology
5. Briefly discuss the Post-Transcriptional Gene Silencing (PTGS) method of
gene silencing giving examples.
NOTES 6. Explain the components and mechanism of RNA interference giving
examples.
7. Discuss the role of RNA-Inducing Silencing Complex (RISC) in gene
silencing.
8. Elaborate on the formation of antisense mRNA and inhibition of gene
expression giving examples.
9. Discuss the formation and regulation of miRNA, siRNA, lncRNAs and
piRNAs.
10. Explain the impact of regulation of Ethylene biosynthesis and post-harvest
fruit management.
11. Write a short note on the applications of RNAi technology in gene silencing
and manipulation of gene expression.

13.8 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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 Plant Genetic

UNIT 14 PLANT GENETIC Engineering

ENGINEERING
NOTES
Structure
14.0 Introduction
14.1 Objectives
14.2 Basics of Plant Genetic Engineering
14.2.1 Ti Plasmid and Agrobacterium Mediated Gene Transfer
14.2.2 CaMV Vector and Its Applications
14.2.3 Direct DNA Delivery Methods
14.2.4 Micro Projectile Bombardment
14.2.5 Microinjection
14.2.6 Electroporation
14.3 Answers to Check Your Progress Questions
14.4 Summary
14.5 Key Words
14.6 Self Assessment Questions and Exercises
14.7 Further Readings

14.0 INTRODUCTION

Genetic engineering, also called genetic modification or genetic manipulation, is

the direct manipulation of an organism’s genes by means of biotechnology. Principally,
it refers to a set of technologies used to change or modify the genetic makeup of
cells, including the transfer of genes within and across the species boundaries for
producing enhanced or novel organisms. New DNA is obtained either by isolating
and copying the genetic material of interest using recombinant DNA methods or
by artificially synthesising the DNA. The first recombinant DNA molecule was
made by Paul Berg in 1972 by combining DNA from the Monkey Virus SV40
with the Lambda Virus. The new DNA can be inserted randomly or can be targeted
to a specific part of the genome.
An organism that is created or produced through the process of genetic
engineering is considered to be Genetically Modified (GM) and the resulting object/
organism is termed as the Genetically Modified Organism (GMO). The first GMO
was a ‘Bacterium’ generated by Herbert Boyer and Stanley Cohen in 1973. Rudolf
Jaenisch created the first GM animal in 1974, when he inserted foreign DNA into
a Mouse. The company Genentech, founded in 1976 was the first company, which
focused on genetic engineering commercially and started the production of Human
Proteins. Genetically engineered ‘Human Insulin’ was produced in 1978 and
‘Insulin-Producing Bacteria’ were commercialised in 1982. Genetically modified
food has been sold since 1994, with the release of the Flavr Savr Tomato. Genetic
engineering has its application in numerous fields, namely medicine, industrial
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Plant Genetic In this unit, you will study about the plant genetic engineering, Ti plasmid,
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CaMV vector, direct DNA delivery methods, micro projectile bombardment,
microinjection and electroporation.

NOTES
14.1 OBJECTIVES

After going through this unit, you will be able to:

• Discuss about the plant genetic engineering and its prospects
• Understand the role of Ti plasmid and the CaMV vector in plants
• Describe the direct DNA delivery methods
• Recognise the various methods of DNA transfer in plants
• Elaborate on micro projectile bombardment
• Explain microinjection and electroporation techniques

14.2 BASICS OF PLANT GENETIC ENGINEERING

Genetic engineering can be specifically used to introduce the precise traits into
plants. The conventional breeding is not replaced but it can enhance the efficiency
of crop upgrading. It enables the regeneration of a new plant from an isolated cell.
IUPAC Definition: According to the IUPAC definition, “The ‘Genetic Engineering’
is the process of inserting new genetic information into existing cells in order to
modify a specific organism for the purpose of changing its characteristics.”
Typically, the dicots are transformed using the Bacterium, Agrobacterium
tumefaciens. Genes are precisely cloned into plant expression vectors that carry
the right and left border sequences. The genes are introduced into the specified
species of plants with the help of a deactivated Ti plasmid whose virulence gene
products allow the genes to be transferred to the plant nucleus where they are
integrated into the ‘Genome’. The monocots are generally transformed through
a biolistic process, using a ‘Gene Gun’.
In both the cases, dicots and monocots, the callus tissue is regenerated on
the specific media that contains an antibiotic or herbicide for transforming. The
exception is transformation of the common wall cress, Arabidopsis thaliana, in
which the tissue culture is not essential. At the post-transcriptional level, the
transgene RNA could be specifically degraded if tagged by a small complementary
RNA molecule. It is often advantageous for plants to express the introduced
transgenes in specific tissues or under specific conditions.
The advent of green revolution was followed by the extensive application of
molecular biology to resolve biotechnological manipulations. Various transgenic
plants have been raised to prevent desiccation, salinity, pathogen infestation and
low temperature tolerance in agricultural fields. Plant genetic engineering involves
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manipulation of gene expression, cloning through recombinant vector and creation Plant Genetic
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of transgenic. Currently large number of transgenic have been created to obtain
plant antibodies and plant antigens. Various pharmacological compounds have
been obtained from their overexpression transgenic, which find their application in
drug yielding. The important aspects of genetic engineering involve successful gene NOTES
transformation into plant cells followed by suitable tissue culture conditions for
screening of recombinant and rearing of transgenic plant tissues. The
Agrobacterium sp. is a Gram-Negative, soil-borne Bacterium, phyto-pathogenic
in nature and commonly causes Crown Gall disease in plants. A part of the tumour
inducing plasmid T DNA gets integrated with the plant chromosome by the means
of site specific recombination. The Ti plasmid is commonly obtained from two
species of the Gram-Negative Bacteria namely Agrobacterium tumefaciens and
Agrobacterium rhizogenes. The growth of the plasmid within the Escherichia
coli is successful at a temperature within 30°C. A specific region in the plasmid
triggers the process of conjugative transfer of a segment of T DNA into the plant
cells. The tumour inducing genes are removed from the Ti plasmid to produce a
deactivated plasmid. The genes present in the T DNA are compatible with the
Eukaryotic expression system and thus are able to translate proteins within the
host cell. This induces the formation of Gall or Tumour in plant tissues. The specific
features of Eukaryotic cell compatibility and virulence factors render the Ti plasmid
to function as an ideal vector for genetic transformation in plants. The T DNA is
flanked by left and right borders of 25 bp each. The right and left border sequences
are not transferred into the plant genome, but they facilitate the process of T DNA
transfer. The protein encoded by VirG activates the Vir gene expression after
binding to the consensus sequence. Agrobacterium cells are capable of establishing
infection within the dicotyledonous members. The monocots are unable to produce
the required signalling components (phenols) essential to establish infection between
the bacteria and the plant cell. The process of integration into the plant genome
occurs by non-specific recombination. The Ti plasmid has been implied as a
successful natural vector for gene transfer in plant cells. The technology of genetic
engineering has enabled the creation of vectors suitable for gene cloning and transfer
via Agrobacterium based gene transfer method. The wild type Ti plasmids are
unsuitable for being used as a vector. The oncogenes present in the T DNA trigger
tumour development in the recipient cells. Co-integrate vectors are modified Ti
plasmid with deletions. The unique method involves sub-cloning of the GOI (Gene
Of Interest) in an intermediate vector preferably raised in Escherichia coli. However,
this type of vectors are generally incapable of replicating in Agrobacterium species
cells independently.
The vector produced from the association of CaMV 35S promoter is effective
in terms of gene transfer and its expression in dicot plants. The entire DNA of
CaMV can be propagated and transmitted through the bacterial hosts by integrating
with plasmid and phage vectors. The method of direct DNA transfer is mediated
by DNA delivery in the cells without using the vectors. This method transfers
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Plant Genetic naked DNA into the cell through various effective and simple methods. The high
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transgene copy number may increase the chances of gene silencing. The method
of gene transfer is always desired to be stable within the plant cell. The transferred
DNA should possess high expression capability and stability in the plant genome.
NOTES Stable gene transfer successfully integrates the GOI into the plant chromosome
and the genetic effect is long lasting.
14.2.1 Ti Plasmid and Agrobacterium Mediated Gene Transfer
The Tumour inducing (Ti) plasmid is commonly used as a vector for genetic
transformation in plants, essentially in dicotyledonous species. The plasmid
possesses naturally occurring genes which facilitate the transfer of a part of T
DNA into the host cells. The Auxin and Opine Synthesis Genes present in the Ti
plasmid promote the formation of Gall or Tumour in plants. The Ti plasmid is
commonly obtained from two species of the Gram-Negative Bacteria namely
Agrobacterium tumefaciens and Agrobacterium rhizogenes. The growth of the
plasmid within the Escherichia coli is successful at a temperature within 30°C.
The various types of opines present in the Ti plasmid are usually octopine, nopaline,
succinamopine and leucinopine.
These molecules are derivatives of Amino Acids called Arginine and Alanine.
The Ti plasmid is essentially around 206,479 nucleotide long with around 196
encoding genes. The plasmid possesses a GC content of 56%. The modified form
of the Ti plasmid is used for gene transformation. A specific region in the plasmid
triggers the process of conjugative transfer of a segment of T DNA into the plant
cells. The tumour inducing genes are removed from the Ti plasmid to produce a
deactivated plasmid. The GOI is inserted in between the left and right borders of
the plasmid and further reintroduced into the Agrobacterium species cells. The
Agrobacteriun species containing the recombinant plasmid is co-cultured with
the plant tissue desired to be transformed with the GOI.
Agrobacterium species is a Gram-Negative, soil-borne Bacterium, phyto-
pathogenic in nature and commonly causes Crown Gall disease in plants. A part of
the tumour inducing plasmid T DNA gets integrated with the plant chromosome
by the means of site specific recombination. The opine synthesizing genes produce
molecules of Carbon source for the bacteria to survive. The process of T DNA
transfer is mediated by the proteins of the virulence genes present in the Ti plasmid.
The virulence proteins mediate the process of transfer, protection from cytoplasmic
nuclease activity and provide stability. The T DNA is then incorporated into the
nuclear genome of the transformed plant cell. The genes present in the T DNA are
compatible with the Eukaryotic expression system and thus are able to translate
proteins within the host cell. This induces the formation of Gall or Tumour in plant
tissues. The specific features of Eukaryotic cell compatibility and virulence factors
render the Ti plasmid to function as an ideal vector for genetic transformation in
plants.
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Structure of Ti Plasmid Plant Genetic
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The Ti plasmids are harboured by the virulent strains of the bacteria and are likely
200-235 kbp long. The Ti plasmid commonly consist of the regions called T DNA,
vir region, origin of replication (ORI), region facilitating conjugative transfer and o- NOTES
cat region (necessary for opine biosynthesis).
T DNA: This region of the DNA is specific in its sequence and is of average length
of 24 kb in size. This part of the plasmid is transferred into the host plant cell and
it gets integrated into the plant DNA, mediated by site specific recombination. The
transfer DNA region of the plasmid is flanked by left and right borders. The T
DNA contains two types of genes, namely Oncogenes and Opine Synthesizing
Genes.
• Oncogenes: The oncogenes are essential for the biosynthesis of Auxins
and Cytokinins which promote the Tissue Proliferation or Gall Formation.
• Opine Biosynthesizing Genes: Various types of opines are utilized by the
bacterial cell and it functions as a source of nutrition.
T-DNA Border Sequences: The T DNA is flanked by left and right borders of
25 bp each. The right border and left border sequences are not transferred into the
plant genome, but they facilitate the process of T DNA transfer. The right border is
precise in its size but the left border may vary between 100 nucleotides. The right
border region has been observed to be associated with the main function of
transferring the T DNA. The left border alone is less important for the transfer of T
DNA.
Virulence (Vir) Genes: The Vir genes facilitate the transfer of the T DNA into
the plant cell. This region of the plasmid contains 35 genes organized into 7 operons
namely, A, B, C, D, E, F and G. The process of gene transfer via the Ti plasmid
occurs through a conjugative process between the host cell and the bacteria. The
VirA eddies for the expression of receptor which binds to the released phenolic
compounds like acetosyringone, syringealdehyde or acetovanillone. The VirB
encodes for the pilus like structure which mediates the DNA transfer between the
host and the bacteria. VirC has been suggested to bind to the overdrive sequence.
The VirD1 and VirD2 encode for endonuclease which cleave the direct repeat
borders of the T-DNA. The VirD encodes for the coupling protein which helps in
the transfer of the DNA. The VirE protein binds to the T DNA and protects it
from the activity of the endonucleases present in the plant cytoplasm. Moreover,
the VirE protein helps to associate the T DNA with lipid molecules, thus facilitating
the transfer complex formation across the conjugation tube. The process of transfer
initiates from the right border of the T DNA. The protein encoded by VirG activates
the Vir gene expression after binding to the consensus sequence. The protein of
the VirG is phosphorylated by the activity of VirA. Self-Instructional
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Plant Genetic Figure 14.1 illustrates the structure of Ti plasmid.
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NOTES

Fig. 14.1 Structure of Ti Plasmid

Process of T DNA Transfer into Plant Cell

The process of T DNA transfer from the bacteria to the plant cell is mediated by
the process of cell-cell recognition and establishment of the conjugation process.
The infection of the bacteria triggers the secretion of specific phenolic compounds
which help in initiating the process of infection. Agrobacterium species cells are
capable of establishing infection within the dicotyledonous members. The monocots
are unable to produce the required signalling components (phenols) necessary to
establish infection between the bacteria and the plant cell. The process of T DNA
transfer is accomplished by the following steps.
A. Recognition and Elicitation of the Vir Gene
The wounded plant tissues release phenolic and sugar compounds which appear
to function for the bacterial-plant cell recognition process. These molecules act as
chemotactic attractants for the Agrobacterium species. The establishment of
infection is accompanied by the recognition of signal by the VirA/VirG2 component
of the signal transduction pathway. The VirA is a bacterial membrane localized
kinase protein which recognizes the presence of acetosyringone in the environment.
The VirA component undergoes auto-phosphorylation and in turn activates the
VirG protein. The VirG protein functions as a transcriptional activator for other
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Vir genes present in the plasmid. The VirG protein has to be phosphorylated in Plant Genetic
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order to exhibit its activity.
B. T DNA Complex Formation
The process of T DNA transfer from the bacteria to the plant cell requires the prior NOTES
formation of T DNA complex within the bacterial cell. The specific region of the
transfer DNA is nicked by endonuclease activity and then undergoes complex
formation. The VirD1/VirD2 border endonucleases recognize the left and right
borders of the T DNA. The VirD2 protein creates a single strand nick on the T
DNA and releases the ss-T DNA. The single stranded T DNA undergoes
attachment of the VirD2 protein at the 5'-end of the displaced end. This results in
the formation of nascent T DNA complex.
C. T DNA Transfer and Integration into Plant Cell
T DNA complex is transferred by facilitated conjugative process mediated by the
pilus like structure. The T DNA complex associates with lipids molecules which
help in transporting the complex across the membrane. The conjugative pilus is
formed by Type-IV secretion system. The Type-IV secretion system is encoded
by the proteins VirA and VirD4. The VirD4 protein functions as a mediator to
facilitate the complex formation between T-DNA-VirD2 components. This complex
further interacts with the VirB encoded pilus required for DNA transfer. The other
components of the Vir gene namely VirE2, VirE3, VirF, VirD5 are capable of
movement through the pilus. These proteins facilitate to maintain the T-DNA-Vir
protein complex in the plant cytoplasm. This results in the formation of a mature
T DNA complex. The process of T DNA transport across the membrane is facilitated
by the Vir genes which enable in the formation of an energy dependent-ATPase
mediated transport. The VirB proteins, i.e., VirB2, VirB5 and VirB7 help in the
formation of the pilus. The VirB2 component is the major form of pilin proteins
which gets cyclised and reused in the cell. VirE2 is the ssDNA binding protein
which coats the T DNA during the process of transfer to the plant cell. VirD2 and
VirE2 and attach to the 5´ end of the T DNA and protect it from the activity of
endonucleases likely to be present in the plant cell cytoplasm. Furthermore, these
two proteins are essential for the trafficking of the T-DNA into the nucleus of the
plant cell. The VirD2 and VirE2 possess Nuclear Localization Signals (NLS) which
primarily guide the T DNA destined for nuclear transport. The VirC2 protein
maintains the efficiency of the transfer by binding to the overdrive enhancer element.
The VIP1, VirF and certain importin family of proteins interact with the T DNA
assembly within the plant cell. The VirF mediated the proteasome mediated
destruction of the coat forming proteins namely VIP1 and VirE2. The ss-T DNA
reaches the nucleus and gets converted into ds-T DNA. The process of integration
into the plant genome occurs by non-specific recombination.
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Plant Genetic
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NOTES

Fig. 14.2 Steps Showing T DNA Transfer and Integration into the Plant Cell

Figure 14.2 illustrates the various steps involved in the T DNA transfer and
integration into the susceptible plant cell, where,
Step 1: Secretion of signalling molecules by wounded tissue.
Step 2: Recognition of signalling molecules by Agrobacterium.
Step 3: Adherence of the bacteria to the plant cell.
Step 4: Activation of Vir genes.
Step 5: Formation of immature T DNA complex.
Step 6: T DNA transfer.
Step 7: Assembly of T complex and nuclear transport.
Step 8: Non-specific recombination and integration of T DNA into plant Genome.
Step 9: Opines can be metabolized by Agrobacterium.
Step 10: Expression of bacterial genes in the plant cell.
Figure 14.3 illustrates the method of Ti plasmid mediated gene transformation in
plants.

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 Plant Genetic
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NOTES

Fig. 14.3 Method of Ti Plasmid Mediated Gene Transformation in Plants

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Plant Genetic Ti Plasmid as a Natural Vector for Plant Based Gene Transfer
Engineering
The Ti plasmid has been implied as a successful natural vector for gene transfer in
plant cells. The technology of genetic engineering has enabled the creation of vectors
NOTES suitable for gene cloning and transfer via Agrobacterium based gene transfer
method. The wild type Ti plasmids are unsuitable for being used as a vector. The
oncogenes present in the T DNA trigger tumour development in the recipient
cells. The oncogene segments are deleted from the natural T DNA and this
disarmed/deactivated plasmid is preferably used as a vector for gene transfer. The
GOI is inserted in the region of the T DNA flanked by RB (Right Border) and LB
(Left Border) and then transferred to the desired plant cell. The pBR322 sequences
have been substituted for the major art of the T DNA of the pTiC58 plasmid.
The RB, LB and nos gene (nopaline synthase (nos) gene) are essentially not
removed from the plasmid. The resulting recombinant construct is termed as
pGV3850. This plasmid is used for gene transfer to plant cells which are devoid
of tumour formation unlike the wild type T-DNA transfer. The recombinant cells
are screened for nopaline production. Fig. 14.4 illustrates the structure of disarmed
Ti plasmid converted into pGV3850 vector.

Fig. 14.4 Structure of Disarmed Ti Plasmid Converted into pGV3850 Vector

The creation of pGV3850 has been an important advancement for gene transfer
by using Ti plasmid as a vector. Opine productions and ocs (octopine
synthase) and nos (nopaline synthase) gene expression in the plasmid are widely
used for screening of recombinant plant tissues. However, the application of Ti
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plasmid possesses certain limitations due to its larger size. Furthermore, there is an Plant Genetic
Engineering
absence of unique restriction enzyme sites present in the Ti plasmid. This renders
it unsuitable for manipulation by restriction digestion with preferred enzymes. The
method of performing enzymatic assays for every transformants has been overcome
NOTES
by the use of selectable marker gene in the construct. The transformed plants are
effectively screened by the application of drug or herbicide resistance gene activated
in the construct.
Co-Integrate and Binary Vectors: Modifications of Ti Plasmid
Co-integrate vectors are modified Ti plasmid with deletions. The unique method
involves subcloning of the GOI in an intermediate vector preferably raised in
Escherichia coli. However, this type of vectors is usually incapable of replicating
in Agrobacterium cells independently. Thus the process of transfer is undertaken
by a process of triparental mating where three bacterial strains which involve an
Escherichia coli strain with a helper plasmid which facilitates to mobilize the
intermediate vector, an Escherichia coli strain carrying the recombinant
intermediate vector and the Agrobacterium tumefaciens cell containing the Ti
plasmid. The conjugation occurring between the two Escherichia coli strains result
in the transfer of the helper plasmid into the cell containing the intermediate plasmid.
This is further followed by the transfer of the intermediate plasmid to the
Agrobacterium cell where it recombines with the Ti plasmid to form a large sized
recombinant co-integrate vector. The resultant recombinant form of the Ti plasmid
is delivered for recombination into plant genome. The stability of the co-integrate
vector depends upon the homology of the two vectors recombined. Ti
plasmid pGV3850 is capable of carrying a segment of the pBR322 sequence in
its T DNA. Figure 14.5 illustrates the construction process of co-integrate vector.

Fig. 14.5 Construction of Co-Integrate Vector

Another important modification of Ti plasmid is the creation of a binary vector.

The mini Ti plasmid with the LB and RB is transferred into an Agrobacterium cell
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Plant Genetic containing the T DNA with the Vir genes being removed (disarmed). The binary
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vectors are capable of utilizing the trans acting Vir genes present in the Ti plasmid
and can also act on any T DNA sequence on the same cell. Interestingly, the GOI
and the Vir genes are present in the separate plasmids. Figure 14.6 illustrates the
NOTES construction process of a binary vector.

Fig. 14.6 Construction of a Binary Vector

14.2.2 CaMV Vector and Its Applications

The Cauliflower Mosaic Virus (CaMV) is a member of Caulimo Virus and belongs
to the Caulimoviridae family. These viruses are para-retroviruses which replicate
by the activity of the enzyme reverse transcriptase. Unlike retroviruses these are
DNA viruses. CaMV mostly infects the members of Brassicaceae and Solanaceae.
Certain strains like D4 and W260 have been reported to cause devastating effects
on plant members. The variations in the strain of infecting virus, host ecotype and
environmental conditions regulated the symptom of the disease. Interestingly, the
transmission of the virus occurs through the activity of the aphid called Myzus
persicae. Followed by infection the virion is transported to the nuclear envelope
of the plant cell. CaMV possesses a double stranded circular DNA of 8 kb which
is interrupted by the nicks. This results due to the prevalence of RNAse H activity
during reverse transcription. Met-tRNA, and two RNA primers are involved in
the process of reverse transcription and is capable of creating the nick. After
entering into the plant cell the DNA is transcribed to form 35S RNA and 19S
RNA fragments.
The promoter functioning to transcribe the 35S RNA is a strong constitutive
promoter which finds application in the gene expression and recombinant vector
construction. This promoter is effective in causing higher levels of gene expression
in dicot plant members in comparison with monocot members. The difference has
been attributed to the variation in the presence of the regulatory factors responsible
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for gene expression. This 35S promoter is also expected to exhibit higher activity Plant Genetic
Engineering
in animal cells. The 35S RNA is a complex structure with 600 nucleotide long
leader sequence and 8 Open Reading Frames (ORF). The mechanism of expression
of the proteins is unique in the sense that ORF VI regulates the translation re-
NOTES
initiation of the major ORFs present on the 35S polycistronic RNA. The eight
ORF functions are as follows:
ORF I – For Movement of Protein
ORF II – Produce Insect Transmission Factor
ORF III – Structural DNA Binding Proteins
ORF IV – Capsid Protein
ORF V – Protease, Expression of Reverse Transcriptase
ORF VI – Translation Activation and Trafficking
ORF VII – Associated Functions
The CaMV promoter (35S) is widely used in the gene expression of transgenic
plants. The promoter is inserted in a different form such that it is active in a wide
range of host types in different environment. The vector produced from the
association of CaMV 35S promoter is effective in terms of gene transfer and its
expression in dicot plants. The foreign DNA is expected to be encapsulated in the
viral protein and the inserted gene should not interfere with the assembly of the
virus. The CaMV vector does not contain any non-coding region. However, the
genes II and IV do not possess any specific function. However, the CaMV DNA
has a limited capacity of expression. The CaMV DNA is unable to accommodate
foreign DNA if its size exceeds that of its normal limit. Exceeding the size limit
inhibits the infectious nature of the virus. There are certain limitations in the packaging
of the Genome and insertion of GOI. However, CaMV DNA can be effectively
packed into nucleosome. The DNA can be effectively transcribed by the activity
of RNA Polymerase II obtained from plant. The entire DNA of CaMV can be
propagated and transmitted through the bacterial hosts by integrating with plasmid
and phage vectors. Insertion of 8-base pair EcoRI linker molecule into the intergenic
region of the cloned CaMV strain CM4-184 DNA has been popularized. This
method does not interfere with the infectious ability. The maximum size of the
DNA that can be propagated through this vector is near to 250 bp. Cloned viral
DNA can be inserted into the plant by rubbing the DNA material on the leaves
with help of an abrasive. The bacterial plasmid used to propagate CaMV in
Escherichia coli should essentially be excised. The virus particles accumulate as
cytoplasmic inclusion in the plant cells. They multiply in a high copy number. Thus
foreign DNA can be easily inserted into the CaMV Genome and delivered to the
plant. The presence of secondary structure in the inserted DNA might also influence
the stability of the viral genome. It might interfere with the transcription and translation
of the virus. Figure 14.7 illustrates the structure of CaMV genome.
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Plant Genetic
Engineering

NOTES

Fig. 14.7 Structure of CaMV Genome

14.2.3 Direct DNA Delivery Methods

The method of direct DNA transfer is mediated by DNA delivery in the cells
without using the vectors. This method transfers naked DNA into the cell through
various effective and simple methods. However, a major limitation of this method
is that it increases the chances of transgene rearrangement. The high transgene
copy number may increase the chances of gene silencing. The method of gene
transfer is always desired to be stable within the plant cell. The transferred DNA
should possess high expression capability and stability in the plant genome. Stable
gene transfer successfully integrates the GOI into the plant chromosome and the
genetic effect is long lasting. The direct transfer by physical method involves the
delivery of DNA/RNA across the breached membrane directly into the cell or
nucleus. The effectiveness of physical transfection is enhanced by its combination
with the Chemical Transfection methods. This improvisation is now widely used
for the successful delivery of recombinant plasmids or viral genomes containing
GOI insert directly into the plant cell. In physical methods of particle bombardment
or electroporation the factor of membrane barrier is omitted. This method also
negates the possibility of cytoplasm mediated DNA instability and lessens the
damage caused. The physical methods appear to be expensive due to the
requirement of specific apparatus.
14.2.4 Micro Projectile Bombardment
This method is one of the effective ways of gene delivery into the plant cell thus
leading to successful creation of transgenic plants. This method has a wide host
range and can be effectively used for both mammalian cells, plants and
microorganisms. This method was initially named Biolistic (Sanford 1988) is a
combinatorial biology with ballistics. Micro-carriers or micro projectiles, Tungsten
or Gold coated DNA are mediated by macro-carriers or macro projectiles. The
later are inserted into the apparatus and moved downwards by pushing and
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rupturing the disc. The presence of the stopping plate restricts the movement of Plant Genetic
Engineering
the macro-carrier but in turn allows the micro-carrier to enter with high speed.
The DNA segments are released into the nucleus and gets integrated into the
genome. The primary explants used for particle bombardment are usually
NOTES
proliferating embryogenic tissues. The tissues are grown in a high osmotic medium
so as to prevent osmotic damage during bombardment. The gene constructs can
be in the form of either circular or linear plasmids. Sorbitol or Mannitol is
preferably used as an Osmoticum during bombardment. This method essentially
facilitates the co-transformation of a multiple transgenes. Study of transient gene
expression and plastid transformation can be effectively performed by this method
of Biolistic.
Gene Integration
1. In the phase prior to integration the vectors are spliced which result in
fragments carrying multiple gene copies.
2. Followed by the earlier step the gene is integrated into the plant genome.
3. The process of bombardment is associated with a multiple number of copies
in the same locus.
4. Various crops among the cereals like maize, wheat and rice have been
transformed by this method.
5. Bt-toxin gene (Bacillus thuringiensis) expressing transgenic maize was the
first crop created by this method.
6. Inert elements like Tungsten or Gold can effectively transport DNA as a
micro-carrier during bombardment. Moreover, the target tissue should
preferably contain more number of actively dividing cells.
7. Exceptionally low amount of DNA may cause failure in successfully
implementing the bombardment method. Alternately, very high amount of
DNA used for transfection results in high number of transgene thus leading
to gene silencing.
8. A suitable condition of humidity, temperature and photoperiodic intensity is
necessary for the tissue following bombardment.
9. Although the method is effective for creating a wide range of transgenic,
multiple copy gene silencing, tissue damage and chimeric expression of
transgene are some of its limitations.
10. Intracellular protection of DNA is less in this method and the sites of
integration are random.
11. Yeast Artificial Chromosomes (YACs; 150 kb) and of BAC plasmid (106
kb) are expected to be successfully transfected by this method.

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Plant Genetic Figure 14.8 illustrates the method of particle bombardment (Biolistic method) of
Engineering
DNA transfection.

NOTES

Fig. 14.8 Method of Particle Bombardment (Biolistic Method) of DNA Transfection

Table 14.1 specifies the list of crops and types of plants used for DNA transfection
by Biolistic method.
Table 14.1 List of Crops and Types of Plants used for
DNA Transfection by Biolistic Method

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14.2.5 Microinjection Plant Genetic
Engineering
The single target cell chosen from embryogenic meristemoid callus tissue or
protoplast is subjected to direct DNA insertion by mechanical method of physical
transfection. This process is termed as microinjection. This method is often used NOTES
for organelle transfer and manipulation of the chromosomal structure.
The method involves the use of a glass micro-capillary injection micro tip of
dimension 0.5-10.0 pm. During the process of gene transfer the micro tip inserts
the DNA into the cytoplasm or nucleus of the cell. Remarkably, the cells are
immobilized on an Agarose embedding matrix using a suction pipette. The Agarose
material used for the process involves low melting temperature. The cells after
being transformed are cultured separately to obtain the screened transformants.
Various species of Tobacco, Petunia, Barley and Mustard have been transformed
by this method. Although the process is very slow, it allows the injection of both
plasmid and chromosomes into the plant cell. The procedure is tedious but ensures
efficiency in the process of DNA delivery into the cell. Investigations of the
macromolecular trafficking has also been studied by this method. Figure 14.9
illustrates how the setup is made for microinjection of DNA into cells.

Fig. 14.9 Setup for Microinjection of DNA into Cells

14.2.6 Electroporation
This method implies the use of high strength electrical impulses which reversibly
permeabilize the cell the cell membrane thus facilitating the transfer of DNA. Intact
plant cells and protoplasts can be transfected by this process. The method involves
incubation of the plant tissue in a specific buffer containing the DNA material desired
to be transferred. Followed by incubation electrical impulse is applied which
permeabilize the membrane. Followed by permeabilization, the DNA enters the
cell and integrates into the Plant Genome. Advancement in the methodology has
facilitated the use of intact callus or other plant tissues to be used for electroporation.
This involves the application of a pre and post electroporation treatment. Rice,
Wheat and Maize have been used for DNA transfer by electroporation.
Among various methods of gene transfer, this method is efficient and cost
effective. The transferred cells do not undergo any change in their physiological
state after undergoing electroporation. Optimising the strength of the electrical
field and use of spermidine increases the efficiency of the transfer. The nature of
the transfecting tissue may affect the amount of DNA transferred into the cells.
The generation of culture from the transformed tissue appears to be difficult in
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Plant Genetic certain cases. Callus tissues have been observed to be more successful than
Engineering
embryos in the process of gene transfer.

Check Your Progress

NOTES
1. Define the term genetic engineering giving example. Also give its IUPAC
definition.
2. What is Tumour inducing (Ti) plasmid?
3. Explain about the Cauliflower Mosaic Virus (CaMV).
4. What is the method of direct DNA transfer?
5. Explain micro projectile bombardment method.
6. What is microinjection?

14.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. Genetic engineering can be specifically used to introduce the precise traits

into plants. The conventional breeding is not replaced but it can enhance
the efficiency of crop upgrading. It enables the regeneration of a new plant
from an isolated cell.
Typically, the dicots are transformed using the Bacterium, Agrobacterium
tumefaciens. Genes are precisely cloned into plant expression vectors that
carry the right and left border sequences. The genes are introduced into the
specified species of plants with the help of a deactivated Ti plasmid whose
virulence gene products allow the genes to be transferred to the plant nucleus
where they are integrated into the ‘Genome’. The monocots are generally
transformed through a biolistic process, using a ‘Gene Gun’.
IUPAC Definition: According to the IUPAC definition, “The ‘Genetic
Engineering’ is the process of inserting new genetic information into existing
cells in order to modify a specific organism for the purpose of changing its
characteristics.”
2. The Tumour inducing (Ti) plasmid is commonly used as a vector for genetic
transformation in plants, essentially in dicotyledonous species. The plasmid
possesses naturally occurring genes which facilitate the transfer of a part of
T DNA into the host cells. The Auxin and Opine Synthesis Genes present in
the Ti plasmid promote the formation of Gall or Tumour in plants. The Ti
plasmid is commonly obtained from two species of the Gram-Negative
Bacteria namely Agrobacterium tumefaciens and Agrobacterium
rhizogenes. The various types of opines present in the Ti plasmid are usually
octopine, nopaline, succinamopine and leucinopine.
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 3. The Cauliflower Mosaic Virus (CaMV) is a member of Caulimo Virus and Plant Genetic
Engineering
belongs to the Caulimoviridae family. These viruses are para-retroviruses
which replicate by the activity of the enzyme reverse transcriptase.
CaMV possesses a double stranded circular DNA of 8 kb which is
NOTES
interrupted by the nicks. This results due to the prevalence of RNAse H
activity during reverse transcription. Met-tRNA, and two RNA primers are
involved in the process of reverse transcription and is capable of creating
the nick. After entering into the plant cell the DNA is transcribed to form
35S RNA and 19S RNA fragments.
4. The method of direct DNA transfer is mediated by DNA delivery in the
cells without using the vectors. This method transfers naked DNA into the
cell through various effective and simple methods. However, a major
limitation of this method is that it increases the chances of transgene
rearrangement. The high transgene copy number may increase the chances
of gene silencing. The method of gene transfer is always desired to be stable
within the plant cell. The transferred DNA should possess high expression
capability and stability in the plant genome.
5. Micro projectile bombardment method is one of the effective ways of gene
delivery into the plant cell thus leading to successful creation of transgenic
plants. This method has a wide host range and can be effectively used for
both mammalian cells, plants and microorganisms. This method was initially
named Biolistic (Sanford 1988) is a combinatorial biology with ballistics.
Micro-carriers or micro projectiles, Tungsten or Gold coated DNA are
mediated by macro-carriers or macro projectiles.
6. The single target cell chosen from embryogenic meristemoid callus tissue or
protoplast is subjected to direct DNA insertion by mechanical method of
physical transfection. This process is termed as microinjection. This method
is often used for organelle transfer and manipulation of the chromosomal
structure.

14.4 SUMMARY

• Genetic engineering can be specifically used to introduce the precise traits

into plants. The conventional breeding is not replaced but it can enhance the
efficiency of crop upgrading. It enables the regeneration of a new plant
from an isolated cell.
• IUPAC Definition: According to the IUPAC definition, “The ‘Genetic
Engineering’ is the process of inserting new genetic information into existing
cells in order to modify a specific organism for the purpose of changing its
characteristics.”
• Typically, the dicots are transformed using the Bacterium, Agrobacterium
tumefaciens. Genes are precisely cloned into plant expression vectors that
carry the right and left border sequences. Self-Instructional
Material 313
Plant Genetic • The genes are introduced into the specified species of plants with the help
Engineering
of a deactivated Ti plasmid whose virulence gene products allow the genes
to be transferred to the plant nucleus where they are integrated into the
‘Genome’.
NOTES
• The monocots are generally transformed through a biolistic process, using
a ‘Gene Gun’.
• In both the cases, dicots and monocots, the callus tissue is regenerated on
the specific media that contains an antibiotic or herbicide for transforming.
• The advent of green revolution was followed by the extensive application
of molecular biology to resolve biotechnological manipulations.
• Various pharmacological compounds have been obtained from their
overexpression transgenic, which find their application in drug yielding. The
important aspects of genetic engineering involve successful gene
transformation into plant cells followed by suitable tissue culture conditions
for screening of recombinant and rearing of transgenic plant tissues.
• The Agrobacterium sp. is a Gram-Negative, soil-borne Bacterium, phyto-
pathogenic in nature and commonly causes Crown Gall disease in plants.
• The Ti plasmid is commonly obtained from two species of the Gram-
Negative Bacteria namely Agrobacterium tumefaciens and Agrobacterium
rhizogenes.
• The growth of the plasmid within the Escherichia coli is successful at a
temperature within 30°C. A specific region in the plasmid triggers the process
of conjugative transfer of a segment of T DNA into the plant cells.
• The specific features of Eukaryotic cell compatibility and virulence factors
render the Ti plasmid to function as an ideal vector for genetic transformation
in plants.
• The Tumour inducing (Ti) plasmid is commonly used as a vector for genetic
transformation in plants, essentially in dicotyledonous species. The plasmid
possesses naturally occurring genes which facilitate the transfer of a part of
T DNA into the host cells.
• The Auxin and Opine Synthesis Genes present in the Ti plasmid promote
the formation of Gall or Tumour in plants.
• The Ti plasmid is commonly obtained from two species of the Gram-
Negative Bacteria namely Agrobacterium tumefaciens and Agrobacterium
rhizogenes. The growth of the plasmid within the Escherichia coli is
successful at a temperature within 30°C.
• The various types of opines present in the Ti plasmid are usually octopine,
nopaline, succinamopine and leucinopine.
• The Ti plasmids are harboured by the virulent strains of the bacteria and are
likely 200-235 kbp long. The Ti plasmid commonly consist of the regions
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 called T DNA, Vir region, origin of replication (ORI), region facilitating Plant Genetic
Engineering
conjugative transfer and o-cat region (necessary for opine biosynthesis).
• The oncogenes are essential for the biosynthesis of Auxins and Cytokinins
which promote the Tissue Proliferation or Gall Formation.
NOTES
• Virulence (Vir) genes facilitate the transfer of the T DNA into the plant cell.
This region of the plasmid contains 35 genes organized into 7 operons namely,
A, B, C, D, E, F and G. The process of gene transfer via the Ti plasmid
occurs through a conjugative process between the host cell and the bacteria.
• The process of T DNA transfer from the bacteria to the plant cell is mediated
by the process of cell-cell recognition and establishment of the conjugation
process.
• The infection of the bacteria triggers the secretion of specific phenolic
compounds which help in initiating the process of infection. Agrobacterium
species cells are capable of establishing infection within the dicotyledonous
members.
• The Cauliflower Mosaic Virus (CaMV) is a member of Caulimo Virus and
belongs to the Caulimoviridae family. These viruses are para-retroviruses
which replicate by the activity of the enzyme reverse transcriptase.
• CaMV possesses a double stranded circular DNA of 8 kb which is
interrupted by the nicks. This results due to the prevalence of RNAse H
activity during reverse transcription. Met-tRNA, and two RNA primers are
involved in the process of reverse transcription and is capable of creating
the nick. After entering into the plant cell the DNA is transcribed to form
35S RNA and 19S RNA fragments.
• The CaMV promoter (35S) is widely used in the gene expression of
transgenic plants. The promoter is inserted in a different form such that it is
active in a wide range of host types in different environment.
• The vector produced from the association of CaMV 35S promoter is effective
in terms of gene transfer and its expression in dicot plants.
• The method of direct DNA transfer is mediated by DNA delivery in the
cells without using the vectors. This method transfers naked DNA into the
cell through various effective and simple methods. The high transgene copy
number may increase the chances of gene silencing.
• The method of gene transfer is always desired to be stable within the plant
cell. The transferred DNA should possess high expression capability and
stability in the plant genome.
• Micro projectile bombardment method is one of the effective ways of gene
delivery into the plant cell thus leading to successful creation of transgenic
plants. This method has a wide host range and can be effectively used for
both mammalian cells, plants and microorganisms.
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Material 315
Plant Genetic • Micro projectile bombardment method was initially named Biolistic (Sanford
Engineering
1988) is a combinatorial biology with ballistics. Micro-carriers or micro
projectiles, Tungsten or Gold coated DNA are mediated by macro-carriers
or macro projectiles.
NOTES
• The single target cell chosen from embryogenic meristemoid callus tissue or
protoplast is subjected to direct DNA insertion by mechanical method of
physical transfection. This process is termed as microinjection. This method
is often used for organelle transfer and manipulation of the chromosomal
structure.
• Electroporation method implies the use of high strength electrical impulses
which reversibly permeabilize the cell the cell membrane thus facilitating the
transfer of DNA. Intact plant cells and protoplasts can be transfected by
this process.
• Electroporation method involves incubation of the plant tissue in a specific
buffer containing the DNA material desired to be transferred.

14.5 KEY WORDS

• Genetic engineering: It is the process of inserting new genetic information

into existing cells in order to modify a specific organism for the purpose of
changing its characteristics.
• Tumour inducing (Ti) plasmid: It is commonly used as a vector for genetic
transformation in plants, essentially in dicotyledonous species. The Ti plasmid
is commonly obtained from two species of the Gram-Negative Bacteria,
namely Agrobacterium tumefaciens and Agrobacterium rhizogenes.
• Oncogenes: The oncogenes are essential for the biosynthesis of Auxins
and Cytokinins which promote the Tissue Proliferation or Gall Formation.
• Virulence (Vir) genes: The Vir genes facilitate the transfer of the T DNA
into the plant cell. This region of the plasmid contains 35 genes organized
into 7 operons namely, A, B, C, D, E, F and G.
• Cauliflower Mosaic Virus (CaMV): It is a member of Caulimo Virus
and belongs to the Caulimoviridae family. These viruses are para-
retroviruses which replicate by the activity of the enzyme reverse
transcriptase.
• Microinjection: The single target cell chosen from embryogenic meristemoid
callus tissue or protoplast is subjected to direct DNA insertion by mechanical
method of physical transfection, this process is termed as microinjection.

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 Plant Genetic
14.6 SELF ASSESSMENT QUESTIONS AND Engineering

EXERCISES

Short Answer Questions NOTES

1. What is the importance of genetic engineering?

2. Explain Tumour inducing (Ti) plasmid.
3. What is T-DNA border sequences?
4. Why the promoter (35S) is widely used in the gene expression of transgenic
plants CaMV?
5. Name the eight ORF functions and their features.
6. Explain direct DNA delivery methods.
7. List the various methods of DNA transfer in plants.
8. What is micro projectile bombardment?
9. Define microinjection and electroporation techniques.
Long Answer Questions
1. Briefly discuss about the scopes and prospects of plant genetic engineering
giving appropriate examples.
2. Discuss the role of Ti plasmid and Agrobacterium mediated gene transfer.
Explain the structure of Ti plasmid with the help of diagram.
3. Elaborate on Ti plasmid as a natural vector for plant based gene transfer.
4. Explain the process of T DNA transfer into plant cell.
5. What is the significance of CaMV vector? Discuss the structure of CaMV
Genome and its applications.
6. Define gene transfection. Explain the direct methods of DNA delivery in
cells.
7. Explain the micro projectile bombardment method with reference to gene
integration.
8. Discuss about microinjection and electroporation processes.

14.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Self-Instructional
Material 317
Plant Genetic Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Engineering
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
NOTES Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
McLennan, Alexander G., Andy D. Bates, Phil C. Turner and Michael R. H. White.
1999. BIOS Instant Notes in Molecular Biology. Oxford (England): BIOS
Scientific Publishers.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.

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318 Material
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