Recombinant DNA Technology

Outline
• Common Terminologies
• Tools of Genetic Engineering
• DNA transfer methods
• Overview of rDNATechnologies
• Applications of recombinant DNA technology
• DNA Library
• Molecular Diagnostic Techniques
• DNA sequencing

By: Teka Obsa (Asst. Prof. of Med. Biochemistry, MSc, BSc in MLT)

By: Teka O. 1
 Common Terminologies

• Recombinant DNA • Vector

• Target gene (DNA) • Host
• Foreign DNA • Molecular techniques
• Genetic engineering • Restriction enzymes
• Cloning • DNA library
• Plasmid • DNA Sequencing
• Bacteriophage • DNA fingerprinting

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 Recombinant DNA Technology

Recombinant DNA (rDNA)

• A form of artificial DNA.
• Prepared by combining DNA from two or more
different sources or species.
• Other names: cloned DNA and chimeric DNA.
• Genetic engineering - artificial manipulation of
organisms’ genetic material.

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 Recombinant DNA Technology

• Refers to several techniques in which

rDNA is involved.
• Also known as is genetic engineering.
• Vectors are used to carry desired gene
(foreign DNA).

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 Tools of Genetic Engineering

1) Enzymes 3) Host cell

• Restriction enzymes • Bacteria
• DNA ligase • Yeasts
• DNA Polymerases • Plant cells
• Reverse transcriptases • Animal cells
2) Vectors
• Plasmid
• Bacteriophage
• Cosmid
• Yeast

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Tools of Genetic Engineering: Enzymes
A. Restriction Enzymes (endonucleases)
• Naturally produced by bacteria.
• Cannot digest host DNA with methylated C.
• Recognize one particular nucleotide sequence in DNA
• Can produce sticky or blunt end DNA.

sticky ends
blunt ends

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 Types of REs
Type I enzymes: Cleave DNA at site remote from
recognition site.
Type II enzymes: Cleave DNA within or at short
specific distances from recognition site.
Type III enzymes: Cleave at sites a short distance
from recognition site.
Type IV enzymes: Target modified DNA.

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 B. Ligase
• Links DNA strands that have double-strand breaks
• Naturally, DNA ligase is used in both DNA
replication and DNA repair.
• DNA ligase has extensive use in molecular biology
laboratories for genetic recombination experiments

ATP DNA ligase

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Fragments of DNA produced by the same restriction
enzyme will spontaneously join by base pairing.

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Tools Genetic of Engineering: Vectors

Vectors - small pieces of DNA used for cloning.

Requirements of the Vector:
1. Self-replication
2. Multiple cloning site
3. Promoter region
4. Selectable marker
5. Proper size

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Vectors: 1. Plasmid
 A small, circular, extrachromosomal dsDNA
 Used as antibiotic resistance
 many copies of plasmid in a bacterium
 replicate independent of the chromosomal DNA.

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Vectors: 2. Bacteriophage
is a virus that can infect bacteria

3. Cosmid
 plasmid + Cos sites
 for binding to bacteriophages
 can carry larger DNA fragments

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 TYPES OF VECTORS

VECTOR kbp

Plasmids 0.01 - 10

Phages 10 - 20

Cosmids 30 - 50

BAC 50 - 250

YAC 500 - 1000

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Tools of Genetic Engineering: Hosts
1. Bacteria
E. coli - used because is easily grown
and its genomics are well understood.
- Gene product is purified from host cells
2. Yeasts - Saccharomyces cerevisiae
– Easily grown and its genomics are known
– May express eukaryotic genes easily
– Continuously secrete the gene product.
– Easily collected and purified

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Tools of Genetic Engineering: Hosts

3. Plant cells and whole plants

– May express eukaryotic genes easily
– Plants are easily grown - produce plants
with new properties.
4. Mammalian cells
– May express eukaryotic genes easily
– Harder to grow
– Medical use

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 DNA transfer methods

Artificial
Natural
1. Physical methods
1. Conjugation 1. Microinjection
2. Bacterial transformation 2. Biolistics transformation
3. Retroviral transduction 2. Chemical methods
• DNA transfer by calcium
phosphate method
• Liposome mediated
transfer
3. Electrical methods
• Electroporation

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 Natural methods of gene transfer
1. Conjugation: Gene transfer between bacterial
cell via pilli.
• Requires the presence of F plasmid.
• The F plasmid consists of 25 genes.

• When the F+ plasmid is integrated within the

bacterial chromosome, the cell is called an Hfr cell.

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 2. Transformation
• The direct uptake of exogenous DNA through the cell
membrane from surroundings.
• It occurs naturally in some spp. of bacteria.
• It can also be effected by artificial treatment in other spp.
• Competent: cell that have undergone this treatment.

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 3.Transduction
• Gene transfer from a donor to a recipient via a
bacteriophage.
• Types: - generalized
- specialized
• Cycle: -Lysogenic cycle
-Lytic cycle

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DNA transfer by artificial methods

Physical methods
1. Microinjection
2. Biolistics transformation
Chemical methods
1. DNA transfer by calcium phosphate
method
2. Liposome mediated transfer
Electrical methods
1. Electroporation

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 Overview of rDNATechnologies
1. Gene of interest (DNA) is isolated (DNA
fragment)
2. Vector and gene of interest are cut by restriction
enzyme
3. A desired gene is inserted into a DNA molecule
(vectors: plasmid, bacteriophage)
4. The vector inserts the DNA into a new cell,
(bacteria, yeast, plant or animal cell)
5. Large quantities of the gene product can be
harvested from the clone.

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Amplifying the recombinant DNA molecule in a
bacterial host

1. Transfection /
transformation

2. Amplify in a suitable
culture medium

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Selection

Isolation

Amplification

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3. Selection, Isolation and Amplification of
Recombinant DNA:
 by specific techniques
 (eg. by antibiotic sensitivity technique)
 Allowed to multiply in a suitable culture.
4. Release of the cloned DNA molecules from the
bacteria
The same RE used for cleaving of DNA is used

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Blue-white screening system

• Vectors: carry LacZ gene and antibiotic

resistant gene
• Hosts lack LacZ gene and antibiotic
resistant gene

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The transformed cells are grown in the presence of:

• ampicillin
• X-gal – substrate for β-galactosidase

– a colourless modified galactose sugar.

– When metabolized by β-galactosidase form
an insoluble product (5-bromo-4-chloro 3-
hydroxyindole) which is bright blue.

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 Results
– Clones lacking the vector will not grow.
– Clones containing the vector without
the new gene will be
– resistant to ampicillin
– able to metabolize X-gal
(blue colonies)
– Clones containing the recombinant
vector will be:
– resistant to ampicillin and
– unable to hydrolyze X-gal
(white colonies).
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 Screening for the desired Gene

• A short piece of labeled DNA (DNA probe) can be

used to identify clones carrying the desired gene.

• Radioactive labeled
• Fluorescent labeled

• Labeled DNA probe ( 32P or fluorescence)

• 5’ *AGGCTTGTACTTTGGCGG 3’

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Applications of recombinant DNA technology

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 Recombinant Pharmaceuticals

• A number of human disorders can caused due to the

absence or malfunction of a normal body protein
• Most of these disorders can be treated by supplying
the patient with the correct version of the protein
• Hence, modern pharmaceutical
manufacturing frequently relies upon
recombinant drugs.

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Recombinant Pharmaceuticals
• Human Insulin
• Human Growth Hormone
• Human blood clotting factors
• Vaccines
• Monoclonal Antibodies
• Interferons
• Antibiotics & other secondary metabolites

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 Human Insulin

• Earliest use of recombinant technology

• Modify E.coli cells to produce insulin
• Performed by Genentech in 1978.
• Prior, bovine and porcine insulin used.
• induced immunogenic reactions.
• many purification and contamination hassles.
• To overcome these problems, researchers inserted
human insulin genes into a suitable vector (E. coli).

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 Application of rDNA technology:
Gene therapy: is an experimental technique that
uses genes to treat or prevent disease.

• Gene therapy was first conceptualized in 1972.

• In the future, this technique may allow doctors

to treat a disorder by inserting a gene into a
patient cells instead of using drugs or surgery.

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Approaches to gene therapy:

 Replacing a mutated gene that causes disease

with a healthy copy of a gene involves:
 Gene knocking out
 Gene knocking in

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 Examples of gene therapy
• Genetic diseases:
• Cystic fibrosis
• Severe combined immunodeficiency disease (SCID)
• Familial hypercholesterolemia
• Hemophilia and Duchenne muscular dystrophy (DMD)
• Acquired diseases:
• Cancer
• cardiovascular diseases
• Alzhemer’s disease
• Parkinson’s disease and AIDS.

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Steps involved in gene therapy

1. Preparation of recombinant DNA

2. Introduction of vector into host cells.
3. Expression of gene in the host.
4. Production of gene product in the host.
5. Deficiency of protein (gene) is corrected.
 Protocols of gene therapy varies from one
disease to another disease.

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Constructing recombinant DNA molecule

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Types of gene therapy

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 IS GENE THERAPY TOTALLY SAFE?

• Although gene therapy is a promising treatment

option for a number of diseases the technique
remains risky and is still under study.
• Gene therapy is currently only being tested for
the treatment of diseases that have no other
cures.

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 Technical Difficulties in Gene Therapy

• Gene delivery: Successful gene delivery is not

easy or predictable, even in single-gene disorders.

• Delivery of genes for cancer therapy may

also be complicated by the disease being
present at several sites.

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Problems with Gene Therapy
Short Lived
• Hard to rapidly integrate therapeutic DNA into
genome
• Would have to have multiple rounds of therapy
Immune Response
• new things introduced leads to immune response
• the gene might be over-expressed (toxicity)
Viral Vectors
• patient could have toxic, immune, inflammatory
response

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 Gene/DNA Library
• A collection of different DNA sequences
from an organism.
• Also called gene banks.
• Cloned into a vector for ease of purification,
storage and analysis.

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Consideration for construction of DNA library

 Size of the gene

 Capacity of the vector
 Molecular tools
 Vectors

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 Uses of gene libraries
• To obtain the sequences of genes for analysis,
amplification, cloning and expression.
• Once the sequence is known probes,
primers, etc. can be synthesized for further
diagnostic work using hybridization reactions,
blots and PCR.
• Knowledge of a gene sequence also offers the
possibility of gene therapy.

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 Types of gene library:
• 2 types of gene library

• Depending upon the source of the DNA used.

1. Genomic library contains DNA fragments representing

the entire genome of an organism.

2. cDNA library contains only complementary DNA

molecules synthesized from mRNA molecules in a cell.

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 Genomic Library:
• Made from nuclear DNA of an organism or species.

• DNA is cut into clonable size pieces as randomly

possible using restriction endonuclease.

• Genomic libraries contain whole genomic fragments

including gene exons and introns, gene promoters,
intragenic DNA, origins of replication, etc.

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Construction of Genomic Libraries

1. Isolation of genomic DNA and vector.

2. Cleavage of genomic DNA and vector by REs.

3. Ligation of fragmented DNA with the vector.

4. Transformation of rDNA in the bacterial cell.

5. Amplification of the rDNA in bacterial cells.

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 Screening of genomic library
• Screening is used to identify the genes of
interest.
• The most common technique is colony
hybridization.
• In the process of library construction, phage
vectors are used then the process of
identification of genes of interest involved is
the plaques hybridization.

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 Colony hybridization
• Is the screening of library used to select bacterial colony
with desired gene.
• Radioactive probe is used to identify a specific sequence of
DNA, RNA.

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 Plaque hybridization
• Used to identify recombinant phage.
• Based on the hybridization of oligonucleotide
probe to target DNA.

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Applications of Genomic Library
1. DNA sequencing projects.
2. Identification of pharmaceutically important genes.
3. Identification of new genes which were silent in
the host.
4. It helps us in understanding the complexity of
genomes.
5. Serving as a source of genomic sequence
6. Studying the function of regulatory sequences in
vitro.
7. Study of genetic mutations in cancer tissues

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 cDNA Library
• DNA molecules synthesized from mRNA
molecules in a cell.
• cDNA is synthesized from a mature mRNA
using reverse transcriptase.
• In eukaryotes, a poly-(A) tail (primer site)
distinguishes mRNA from tRNA and rRNA.

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cDNA Construction

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Screening a cDNA or Genomic Library
1. Immobilize members of the library onto a nylon
membrane and denature them.
2. Prepare single-stranded radio-labelled probe
3. Hybridize the probe to the library of clones
4. Wash the excess probe and expose to X-ray film
5. Isolate the positive clone and analyze

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 cDNA Library uses

• Useful for subsequently isolating the gene that

codes for mRNA.

• To express eukaryotic genes in prokaryotes.

• Useful in reverse genetics where the additional

genomic information is of less use

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cDNA Library vs Genomic DNA Library

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 MOLECULAR DIAGNOSTIC TECHNIQUES
• Common methods used in molecular biology,
biochemistry, genetics and biophysics.
• Generally involve manipulation and analysis of
DNA, RNA and Proteins.
• Used in diagnostic pathology
• neoplastic disorders
• infectious diseases
• inherited conditions and
• Forensic science and identity determination.

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MOLECULAR DIAGNOSTIC TECHNIQUES
• Polymerase chain reaction
• Electrophoresis
• Blotting techniques
• Southern Blot
• Northern Blot
• Western Blot
• In situ hybridization
• Dot blot hybridization
• Microarray

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 Polymerase Chain Reaction (PCR)
• Developed by Kary Mullis in 1984.
• in vitro method for DNA amplification
• much faster
• more sensitive method than cloning.
• very little DNA sample is sufficient
• can only amplify short segments of DNA

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Components required to perform PCR

1) Target DNA
2) A heat-stable DNA
Polymerase (Taq Polymerase)
3) All four nucleotide
triphosphates
4) Buffers
5) Short primers (2ssDNA)
6) Thin walled tubes
7) Thermal cycler

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 Primer design
• Length should be between 18 to 25 bases
• Always specified 5’ to 3’
• Should have 40 - 60 %GC content
• 3’ should end with C or G.
• Should not have complementary regions

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 PRIMERS

• 2 set of primers
• Synthetically
produced.
• 40-60% GC content
preferred.

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 3 steps of PCR
1.Denaturation at 95ºC (30-60s)
2.Annealing (hybridization)- at 60-65 º C (30-
60s)
3.Extension of DNA at 72 ºC (1min)

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 Advantages of PCR

1. Very little DNA sample is required

2. Amplification time is very short.
3. Amplification rate is high.

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 Uses of PCR

1. Diagnostic uses
used to quickly detect microbial
infections, when the number of microbes is
less in the sample.
Examples :
Diagnosis of tuberculosis (TB, HIV).

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2. Prenatal diagnosis of genetic disorders
Sections of genes, having particular

mutations known to cause a disease are

 Amplified
 Sequenced
 Diagnosed
Example: Detection of Sickle cell anemia (HbS)

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3. Forensic Uses:
Samples used : Blood, saliva, semen, hair
Obtained from : a victim or suspect
Volume of the sample: is insufficient
Sample PCR Amplification of DNA

Amplified DNA DNA analytical techniques

i.e., DNA fingerprinting

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Types of PCR
• Conventional PCR
• Reverse transcriptase PCR (RT-PCR)
• Real - time PCR
• Quantitative real-time PCR (q-RT-PCR)
• Multiplex PCR
• Nested PCR, ETC.

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 Applications of PCR

• Diagnosis of infectious agents, including HIV, hepatitis,

malaria, anthrax,
• Prognosis of patient (predicts response or resistance to
therapy).
• Analysis of mutations that occur in many genetic
diseases
• Forensic (DNA fingerprinting)
• Cloning of gene for further study
• Human Genome Sequencing
• Evolutionary biology (Paleontology)

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 Cloning VS PCR

Cloning PCR

• in vivo method using • an in vitro method using

bacteria DNA polymerase
• used to amplify longer
segments of DNA • shorter segments of DNA
can be amplified
• suitable for large-scale
• shorter time for amplifying
protein production DNA fragments

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ELECTROPHORESIS

OUTLINE:
•Definition
•General Principle
•Factors Affecting Electrophoresis
Gel Electrophoresis
•Applications

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 GENERAL PRINCIPLE
• Electrophoresis: Differential movement or migration
of charged molecules (ions) in solution, with response
to an electrical current.
• Separation of molecules according to size and/or
charge.
• Negatively charged molecules (anions) will be move
towards anode.
• Positively charged molecules (cations) will move
towards cathode.
Anode

Cathode
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 Basic Concepts

• As an analytical tool,
electrophoresis is simple, rapid
and highly sensitive.
• Rate of migration depends on:
 Molecular charge (net charge)
Molecular shape and size
Strength of the electrical field,
Ionic strength, viscosity, and
temperature of the medium.

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 ELECTRIC FIELD
• Electric current is carried by buffers
– Buffers keep the pH and charge surrounding
analyte constant.
• Effects of the electric field on the sample:
– In electrophoresis either current, voltage, or
power, is always held constant.
– Higher voltage causes greater migration speed.
– Also leads to generation of heat.
• May denature protein sample and destroy gel matrix
• Also mixes samples through convection of buffer
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 FACTORS AFFECTING ELECTROPHORESIS

The speed and direction of a moving charged particle influenced by

Internal Factors External Factors

• Voltage & Temperature

• Charge of the molecule in voltage & temperature
in charge = “faster speed” = speed = heat and
• Size and Shape leads to protein denaturation
in size = “slower speed" • Buffer pH
pH determines net charge of the
protein, hence direction of migration.
• Supporting medium
- Protein interaction slows speed
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 TYPES OF ELECTROPHORESIS
Electrophoresis

Moving Boundary Electrophoresis Zone Electrophoresis

• Capillary Electrophoresis (CE) • Paper Electrophoresis
Used to separate:
• Capillary Gel Electrophoresis
 Proteins
 Peptides & Amino acids • Gel Electrophoresis
 Inorganic ions - Agarose gel (DNA & Protein)
- Polyacrylamide gel (PAGE)
 Organic bases & acids
- SDS-PAGE (Protein)
 Whole cells
 Nucleic acids

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 GEL ELECTROPHORESIS
Separation of a mixture of charged molecules
• A thin layer or zone of the macromolecule solution is electrophoresed
through solid support matrix (Gel).
• Charged molecules are separated based on their charge and size.

Positive
Molecules

Charge Size
Separation Separation

Mixture of
Charged
Negative
Molecules
Molecules

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 GEL ELECTROPHORESIS

Gel Electrophoresis: Supporting medium is Gel

Gels are composed of polymers of sugars (Agarose or
Polyacrylamide)
• Agarose – a complex sugar chain from red seaweed.
• It has a large pore size good for separating large
molecules.
• Polyacrylamide – chain of acrylamide molecules.
• It has a small pore size good for separating small
molecules.

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 ELECTROPHORESIS OF DNA
Agarose Gel Polyacrylamide (PAGE)
• Separates fragments based on • Used to obtain high resolution
mass and charge. separations.
• They have large pore sizes and • They have small pore size gel
are used for separating larger and are used to separate most
DNA molecules (RFLP proteins and small nucleotides.
Analysis or RNA separation). • Used for the separation of
• Also used to separate large smaller DNA molecules (STR
proteins and protein complexes. analysis and DNA sequence
analysis.
• Typically resolve 200 bp-20 kbp
• Separates fragments < 200 bp

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Gel Electrophoresis Apparatus and Types
• Horizontal Gel Units (“Submarine Gels”)
– Agarosegels
– Most DNAandRNA gels
– Continuous buffer system

• Vertical Gel Units

– Polyacrylamidegels (PAGE)
– Typicallysequencinggels
– Discontinuous buffer system

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 APPLICATIONS of ELECTROPHORESIS

• Used to study the properties of a single charged species or

mixtures of molecules.
• Used to separate organic bases, acids and inorganic ions.
• Used to identify amino acids, peptides and proteins.
• Used to separate very large proteins, nucleic acids and
nucleoproteins etc.
• Used in Clinical Laboratory to separate proteins from each
other
– Proteins analysis in body fluids: Serum, Urine, CSF
– Proteins in erythrocytes: Hemoglobin
– Nucleic acids: DNA, RNA

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 APPLICATIONS of ELECTROPHORESIS

• Agarose Gel electrophoresis is used to visualize:

– Genomic DNA
– RNA
– PCR products
– Plasmids
– Restriction enzyme digest products

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 BLOTTING TECHNIQUES
• The analytical techniques used for the identification
of specific DNA, RNA or a Protein from thousands
of each.

TYPES
• Southern Blot
• Northern Blot
• Western Blot
• Eastern blot

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 SOUTHERN BLOTTING
• Professor Sir Edwin
Southern, developed
this method in 1975.
• Principle: Based on
specific base pairing
rule of complementary
nucleic acid strands.

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SOUTHERN BLOTTING

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 NORTHERN BLOTTING

• Developed by James Alwine and George Stark in 1979.

Principle: RNA – DNA hybridization technique.

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 WESTERN BLOTTING

• Principle: Antigen-antibody interaction.

• Antigen – protein of interest

• Antibody – probe

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WESTERN BLOTTING

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DNA Fingerprinting

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 DNA Fingerprinting is a way to identify a
certain individual, rather than simply
identifying a species or a particular trait.
 A technique used by scientists to distinguish
between individuals of the same species
using only samples of their DNA.

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DNA Finger printing procedure

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 Theprocess of DNA
fingerprinting was invented
by Alec Jeffreys in 1985.

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 Biological sample used for DNA fingerprinting

 Blood
 Hair
 Saliva
 Semen
 Body tissue cells
 Vaginal
cells outside of
a condom

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 Diagnosis of Inherited Disorders
 Helps diagnose disorders in both
prenatal and newborn babies
 Disorders may include cystic
fibrosis, hemophilia, Huntington’s
disease, familial Alzheimer’s, sickle
cell anemia, thalassemia, etc.

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 Crime
 Forensic science is the use of
scientific knowledge in legal
situations.
 The DNA profile of each
individual is highly specific.
 The chances of two people
having exactly the same DNA
profile is 30,000 million to 1
(except for identical twins).

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 The pattern of the DNA profile is compared with
those of the victim and the suspect.
 If the profile matches the suspect it provides strong
evidence that the suspect was present at the crime scene.
 Note: it does not prove that he committed the crime.
 If the profile doesn’t match the suspect, then that
suspect may be eliminated from the enquiry.

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 A violent murder occurred.

 The forensics team retrieved a blood sample

from the crime scene.
 They prepared DNA profiles of the blood
sample, the victim and suspects as follows:

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Were the suspects at the crime scene?

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 Solving Medical Problems

 DNA profiles can be used to determine

whether a particular person is the parent
of a child.
 Thisinformation can be used in
 Paternity/maternity suits
 Inheritance cases
 Immigration cases

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 By comparing the DNA profile of a mother
and her child
 identify DNA fragments in the child, which are
absent from the mother and must therefore
have been inherited from the biological father.

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Who is the father of the child?

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 Some other uses of DNA fingerprinting
 Identification of carcass of tissues
 Detection of somatic mutations or cancer
 Pathogen identification
 Detection of loci controlling quantitative traits
or disease resistance
 Sex determination
 Paternity/maternity disputes
 Individual identification (Military personnel)

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 Genetic markers
• Biological features that are determined by allelic forms
of genes or genetic loci.
• Represent genetic variation between individual
organisms or species.
• Do not represent the target genes themselves, but act as
‘signs’ or ‘flags’
• Located in close proximity to genes (gene tags).
• Can be transmitted from one generation to another.

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Classification of genetic markers
Classical markers
• Morphological markers
• Cytological markers
• Biochemical markers
DNA markers

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 DNA markers
• DNA marker is a small region of DNA sequence
showing polymorphism.
refers to a specific DNA variation between
individuals.
These different DNA or genetic variants are
known as alleles.
DNA marker testing or genotyping determines
which alleles an animal is carrying for a DNA
marker(s).

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APPLICATIONS OF MOLECULAR DIAGNOSTIC TECHNIQUES

• Diagnosis of diseases
• Identification of new organism
• Determination of identity
• Abundant protein production
• Gene therapy
• Industrial and agricultural applications
• Transgenesis
• Evolution

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DNA Sequencing

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 Historical Timeline
1870 – Friedrich Miescher discovered DNA
1940 - Avery: Proposed DNA as ‘Genetic Material’
1953 – Watson & Crick “double helical structure”
1970 - Wu: Sequences λ Cohesive End DNA
1977 – Sanger: Dideoxy Chain Termination
1977 – Gilbert: Chemical Degradation
1986 – Partial Automation
1990 – Cycle Sequencing, Improved Sequencing Enzymes,
Improved fluorescent detection schemes
2002 – NGS: 454 Pyrosequencing
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 DNA sequencing
 The process of determining the order of nucleotides along a
DNA strand.
 All the information required for the growth and development
of an organism is encoded in the DNA of its genome.
 So, DNA sequencing is fundamental to genome analysis and
understanding of the biological processes in general.

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1. Sanger; Chain Termination Sequencing method
2. Maxam and Gilbert; Chemical Sequencing method
3. Next generation sequencing methods; High
throughout put method

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 Frederick Sanger

• British biochemist
• Won the Nobel Prize twice
• 1958 - structure of proteins, insulin
• 1980 – DNA sequencing.

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 Sanger’s Sequencing method
• Chain termination / Dideoxy method
• It is PCR based method
• A modified DNA replication reaction
• Growing chains are terminated by ddNTPs.

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 Principle of Sanger method
• Based on the selective incorporation of chain-
terminating ddNTs by DNA polymerase during in
vitro DNA replication.
• The sequence of a single-stranded DNA molecule
is determined by enzymatic synthesis of
complementary polynucleotide chains.

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Requirements
• Template of DNA to be sequenced
• PCR
• DNA Polymerase
• Primer
• All 4 dNTP
• All 4 ddNTP

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 Sanger Sequencing: Process

1. Get enough quantity of DNA (run PCR)

2. Aliqot DNA into four different tubes
3. Prepare PCR reaction mix as below:
•Primer, Taq polymerase, template (ssDNA), dNTPS (All)
and ddNTPs (ddATP, ddGTP,ddCTP & ddTTP respectively).

4. Run PCR
5. Perform Gel Electrophoresis
6. Interpret results

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Steps of Dideoxy Sequencing
1. A primer is annealed to a single-stranded section of DNA
2. DNA- primer mixture is put into 4 separate tubes with DNA
polymerase and a solution of ddNTPs at a concentration of 100
times lower than the dNTP concentration.
3. DNA Pol uses dNTPs to extend the DNA
4. ddNTPs are put together randomly, resulting in different lengths
of fragments
5. Fragments that are from each of the reactions are denatured
and separated by size using gel electrophroesis
6. The gel is used to visually detect the DNA fragments. The
fragments are to be read from bottom to top (5’ - end to 3’-
end), (complementary sequence).
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Example of Sanger sequencing

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e.g. 2

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Sanger sequencing practice
• Sample DNA: AGCTTCAGTC
• What are the fragments produced by Sanger
method?
• What is the sequence read on the gel?

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Maxam–Gilbert sequencing method
Chemical method

Developed by Allan Maxam and Walter Gilbert

in 1976–1977.

Principle: 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.
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Maxam Gilbert Sequencing: Process Summarized

1. Label 5’- end of DNA

2. Aliquot DNA sample in 4 tubes
3. Perform base modification reaction
4. Perform cleavage reaction
5. Perform gel Electrophoresis
6. Perform autoradiography
7. Interpret results
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I. Chemical Modification of DNA; radioactive
labeling at one 5' end of the DNA (typically
by a kinase reaction using gamma-32P ATP)

II. Purification of the DNA fragment to be sequenced

III. Chemical treatment generates breaks in DNA
IV.Run on the gel

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Chemical Treatment

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 Example: 5-CTACGTA-3

32PO45-CTACGTA-3 (5’- end radiolabeled)

Tube 1: A+G Tube 3: C

32PO45-
32PO45-CT
32PO45-CTA
32PO45-CTAC

32PO45-CTACGT Tube 4: T+C:

32PO45-
Tube 2: G
32PO45-C
32PO45-CTAC
32PO45-CTA

32PO45-CTACG

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Gel electrophoresis

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Interpretation (Sequencing)

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Next-generation sequencing

• Pyrosequencing
• Sequencing by synthesis
• Sequencing by ligation
• Ion semiconductor sequencing

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The genomic strand is fragmented, and the bases in each
fragment are identified by emitted signals when the fragments
are ligated to a template strand.

The NGS method combines the techniques developed

in Sanger sequencing to process millions of reactions in
parallel, resulting in very high speed and throughput at a
reduced cost.

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 Three general steps in NGS

1. Library preparation: libraries are created using random

fragmentation of DNA, followed by ligation with
custom linkers
2. Amplification: the library is amplified using
clonal amplification methods and PCR
3. Sequencing: DNA is sequenced using one of
several different approaches

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 Pyrosequencing
• Basic principle
• Relationship between pyrophosphate and
light emission.
• Functions of sulfurylase, luciferase and
apyrase
• Interpretation

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 Principle
• A DNA sequencing technique based on sequencing by
synthesis.
• Nucleotides are added iteratively to the reaction and in
case of incorporation, pyrophosphate (PPi) is released.
• PPi triggers a series of reactions resulting in production
of light, which is proportional to the amount of DNA
and number of incorporated nucleotides.
• Light generated by luciferase is detected and recorded
by a detector system (CCD) in the form of a peak signal.

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 General procedure
• DNA fragment is amplified with PCR.
• Adapter binding to ends of DNA fragments
• One of the two stands used is labeled with biotin
• Streptavidin-bound beads are taken.
• Amplified DNA is coupled with Streptavidin-bound
beads and denatured with NaOH.
• Washing step: The biotin labeled chain remained bound to
beads.
• Template is pipetted onto a microtiter plate and reaction
mixtures are added.
• NB: instead of dATP, deoxyadenosine alph-thiotriphosphate
(dATPαS) is added.

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Biochemistry of
Pyrosequencing

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• The light intensity is proportional the amount
nucleotides added (2,3,.. fold) as high as normal.
• If no nucleotide is inserted, it remains pitch black in
the well.

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Application of DNA sequencing

• Deciphering “code of life”

• Identification of mutations
• Evolutionary relationships
• Gene identity and mapping
• Gene expression
• Typing microorganisms

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 Safety Issues and Ethics of rDNA

• Ethical questions
– Should employers and insurance companies have access
to a person’s genetic records?
– Will some people be targeted for either breeding or
sterilization?
– Will genetic counseling be available to everyone

• GMO - Genetically modified crops must be safe for

consumption and for release in the environment.

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 Safety Issues and Ethics of rDNA

Accidental release - strict safety standards are

used to avoid it
– Some microbes used in recombinant DNA
cloning have been altered so that they cannot
survive outside the laboratory.
– Microorganisms intended for use in the
environment may be modified to contain
suicide genes (do not persist in the
environment).

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 ETHICAL IMPLICATIONS

• Laboratory genetic manipulations may

create certain new pathogens.
• Biological contamination.
• Detect disease susceptibility in
clinically disease-free individuals.
• Human Genome Project is addressing
these issues through its ELSI component.

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