UNIT – II (Part-I)

• Recombinant DNA technology, which is also called gene cloning or molecular cloning, is a
general term that encompasses a number of experimental protocols leading to the transfer of
genetic information (DNA) from one organism to another.

Steps involved in Recombinant DNA technology:

8. Storing the rDNA into genomic library (full chromosomal DNA) or cDNA library (represents the
mRNA of the transformed cell).

9. Amplification of the stored sample for further analysis when the need arrives.
Restriction modification systems (Restriction enzymes/ endonucleases):
Types I, II, III :- Daniel Nathans, Werner Arber and Hamilton Smith

• Recombinant DNA technology would not exist without the availability of enzymes that recognize
specific double-stranded DNA sequences and cleave the DNA in both strands at these sequences
(restriction enzymes, or restriction endonucleases).
• Nucleases that cut nucleic acid molecules internally are endonucleases, and those that degrade
from the ends of nucleic acids are exonucleases.

The foundations of rDNA technology were laid by the discovery of restriction enzymes. These
enzymes exist in many bacteria where they function as a part of a defence mechanism called the
Restriction-Modification System. This System consists of two components:

1. A restriction enzyme that selectively recognises a specific DNA sequence and digests any DNA
fragment containing that sequence. The term restriction is derived from the ability of these
enzymes to restrict the propagation of foreign DNA (e.g. Viral/phage DNA) in a bacterium.
2. A modification enzyme that adds a methyl group to one or two cytosine residues within the
sequence recognised by the enzyme. Once a base is modified by methylation, the sequence
cannot be digested.

• It is thus obvious that the Restriction-Modification enzyme system within a given bacterium
protects its DNA from digestion by methylation but can digest foreign DNA which is not
protected by similar methylation.

Feature Type I Type II Type III

Recognition Bipartite, two different Palindromic Asymmetric
Sequence sequences
Recognition Long, usually >20 base Short, typically 4-8 Short, typically 5-7
Sequence Length pairs base pairs base pairs
Methylation Hemi-methylated DNA Usually not Sensitive to DNA
Specificity influenced by methylation status
methylation
Enzyme Complex Multimeric with Typically Multimeric with
Composition multiple subunits homodimeric or multiple subunits
homotetrameric
Cleavage Site Random, 1000bp away At or near the 24-26 bp
from recognition site recognition site downstream from
the recognition site
Cofactor ATP-dependent, Mg2+, Generally ATP-dependent,
Requirement S-adenosylmethionine independent of Mg2+(SAM)
(SAM) ATP, Mg2+
Modification Yes, contains a Lacks modification Yes, contains a
Enzyme Activity modification activity modification
methyltransferase methyltransferase
activity activity
 DNA Translocation Yes, translocates along Generally Yes, ATP-dependent
DNA after recognition stationary at translocation before
recognition site cleavage
DNA Cleavage Induces double-strand Precise double- Complex mechanism
Mechanism breaks randomly strand cleavage at involving ATP-
specific sites dependent
translocation and
cleavage
DNA Methylation Prevents cleavage at Usually not May enhance or
Effect on Cleavage recognition site affected inhibit cleavage
depending on
methylation status
Site of Action Cytoplasmic and Mainly cytoplasmic Mainly cytoplasmic
nuclear
Examples EcoKI, EcoR124I, EcoAI EcoRI, HindIII, EcoP15I, EcoPI, PstI
EcoRV

Applications DNA methylation Molecular biology DNA methylation

studies, epigenetics techniques like studies, epigenetics
research cloning research

Nomenclature of restriction enzymes:

The naming protocol for these enzymes is the same as that for EcoRI:

• The genus is the capitalized letter (first letter), here, ‘E’

• The first two letters of the species name are in lowercase letters after the first letter of generic
name, here, ‘co’.
• The strain designation is occasionally added to the name, such as ‘R’ in EcoRI, or the serotype of
the source bacterium is sometimes noted, such as ‘d’ in HindIII.
• The Roman numerals, here, ‘I’ are used to designate the order of characterization of different
restriction endonucleases from the same organism. For example, HpaI and HpaII are the first and
second type II restriction endonucleases that were isolated from Haemophilus parainfluenzae.
Applications of Type II restriction enzymes in genetic engineering:

Type II restriction enzymes play a crucial role in genetic engineering due to their ability to recognize
specific DNA sequences and cleave DNA at or near these sequences. Here are some key applications
of Type II restriction enzymes in genetic engineering:

1. Cloning of DNA Fragments: Type II restriction enzymes are extensively used in the cloning of
DNA fragments. They cleave the DNA at specific recognition sites, generating fragments with
compatible cohesive (sticky) ends. These cohesive ends allow the DNA fragments to be easily
ligated into plasmid vectors or other cloning vectors.
2. Creation of Recombinant DNA: By using Type II restriction enzymes to cut both a plasmid vector
and a foreign DNA fragment at specific sites, scientists can create recombinant DNA molecules.
The foreign DNA can be inserted into the vector, leading to the formation of a hybrid DNA
molecule.
3. Restriction Mapping: Type II restriction enzymes are employed in restriction mapping to analyze
the arrangement of restriction sites within a DNA molecule. By digesting DNA with different
enzymes, scientists can create a map of the positions of specific recognition sequences.
4. Site-Directed Mutagenesis: Researchers use Type II restriction enzymes to introduce specific
mutations at desired locations in a DNA sequence. After cleaving the DNA at a particular site,
synthetic oligonucleotides containing the desired mutation can be ligated into the cut DNA,
leading to site-directed mutagenesis.
5. Polymerase Chain Reaction (PCR) Analysis: Type II restriction enzymes are used in conjunction
with PCR to analyse the success of the PCR reaction. After amplifying a DNA fragment, it can be
digested with a specific enzyme to confirm the presence of the expected sequence.
6. Gene Expression Studies: In studying gene expression, Type II restriction enzymes can be
employed to cleave DNA at specific sites within regulatory regions. This allows researchers to
investigate the impact of alterations in these regions on gene expression.
7. DNA Sequencing: Type II restriction enzymes are utilized in DNA sequencing methodologies.
They can be used to generate a series of nested fragments for the Sanger sequencing method,
providing information about the order of nucleotides in a DNA sequence.
8. Molecular Cloning and Recombinant Protein Production: Type II restriction enzymes are
essential in molecular cloning for the creation of recombinant proteins. They are used to insert a
gene of interest into an expression vector, allowing the production of the desired protein in a
host organism.
9. Genomic Analysis and Genotyping: Type II restriction enzymes are used in various genomic
studies, including genotyping and DNA fingerprinting. By cleaving DNA at specific sites,
researchers can analyse genetic variations and study polymorphisms within populations.
10. Construction of DNA Libraries: Type II restriction enzymes are employed in the construction of
DNA libraries. By digesting genomic DNA with these enzymes, researchers can create fragments
suitable for cloning into vectors, leading to the generation of comprehensive libraries for further
study.
Mode of action of restriction enzymes:
The mode of action of restriction enzymes involves specific recognition of DNA sequences, binding to
the recognition site and subsequent cleavage of the DNA at or near these recognition sites.

1. Recognition of Specific DNA Sequences (Non-specific binding) : Restriction enzymes first

establish a non-specific contact with DNA and bind to it in the form of dimer. They recognize
specific restriction sites by hopping (long distances) or sliding (short distances) on the DNA
strand.

2. Binding to the Recognition Site (specific binding): The restriction enzyme locates and binds to
its specific recognition site on the DNA. Various conformational changes occur in the enzyme as
well as the DNA. These conformational changes activate the catalytic center. The binding is
highly specific, as the enzyme interacts with the nucleotide sequence through hydrogen bonding
and other molecular interactions.

3. Catalysis: After binding to their specific recognition sites, the restriction enzymes fascilitates the
catalysis of the phosphodiester bonds. The enzyme makes two incisions, one on each strand
(sugar phosphate backbones) of the double helix without damaging the nitrogenous bases. This
catalysis happens in three ways:

a. 5’ overhangs: The enzyme cuts asymmetrically within the recognition site such that a
short single-stranded segment extends from the 5’ends. Eg. BamHI.

b. 3’ overhangs: asymmetrical cutting within the recognition site, but the result is a single-
stranded overhang from the two 3′ ends. Kpnl cuts in this manner.

5’-3’ overhangs are also called sticky or cohesive or staggered (uneven) ends as they can
bind easily to the fragments produced by the same enzyme because of complementarity
between the single stranded regions of the fragments produced.
c. Blunt ends: Enzymes that cut at precisely opposite sites in the two strands of DNA
generate blunt ends without overhangs. Smal is an example of an enzyme that generates
blunt ends.
4. After catalysis the enzymes dissociates and fragments of DNA are obtained, the fragments
having overhangs can join with the help of DNA ligase (T4 ligase). The blunt ended fragments can
also be joined together with the help of DNA ligase, called blunt end ligation, matching of
complementary ends aren’t required for ligation and the process is difficult as there’s no
supporting overhang, blunt ends require 10 to 100 times more T4 ligase than sticky ends for
ligation.
Star Activity of Restriction Enzymes:
Star activity refers to a phenomenon where an enzyme, typically a restriction endonuclease, exhibits
relaxed specificity (infidelity towards its primary restriction site) during DNA cleavage. Instead of
recognizing and cleaving its primary restriction site with high fidelity, the enzyme may also cleave
sequences that differ slightly from the primary restriction site. This non-specific cleavage activity is
termed "star activity."

• Causes of Star Activity:

o Altered Recognition Sequence: Star activity can result from changes in the recognition
sequence due to mutations or modifications in the DNA sequence.
o Suboptimal Reaction Conditions: Deviations from optimal reaction conditions (e.g.,
variations in pH, temperature, or salt concentration) can lead to star activity.
o Presence of Co-factors: Some restriction enzymes are sensitive to the presence of
specific cofactors or metal ions. Changes in cofactor concentration can influence enzyme
specificity.
o DNA Methylation: Methylation of the recognition sequence can impact the enzyme's
ability to bind and cleave DNA. Some enzymes are sensitive to the methylation state of
their recognition sites.

• Consequences of Star Activity:

o Non-specific Cleavage: Star activity can result in cleavage at sites that deviate from the
canonical recognition sequence, leading to non-specific DNA cleavage.
o Reduced Fidelity: The fidelity of DNA digestion is compromised, making it challenging to
achieve precise and predictable DNA cleavage.
o Impaired Cloning and Manipulation: In molecular biology applications like DNA cloning
and genetic engineering, star activity can lead to undesirable outcomes, such as
incorrect insertions or incomplete digestion.

• Prevention and Mitigation Strategies:

o Optimization of Reaction Conditions: Ensure that the reaction conditions, including pH,
temperature, and salt concentration, are optimized according to the recommendations
of the enzyme manufacturer.
o Use of Correct Buffer: Use the recommended reaction buffer provided by the enzyme
manufacturer, as different buffers can have a significant impact on enzyme specificity.
o Limited Incubation Time: Minimize the incubation time to reduce the likelihood of star
activity. Shorter incubation times may help maintain the enzyme's specificity.
o Use of Dam-/Dcm-Methylase-Free DNA: If sensitivity to DNA methylation is a concern,
use DNA that is free from Dam (adenine-specific methyltransferase) and Dcm (cytosine-
specific methyltransferase) methylation.
o Enzyme Dilution: Diluting the restriction enzyme can sometimes reduce star activity
while maintaining sufficient activity for specific cleavage.
o Purification of DNA: Ensure high-quality, purified DNA free from contaminants that may
influence enzyme activity.
• Detection of Star Activity:

o Agarose Gel Electrophoresis: Analyze digested DNA fragments on an agarose gel to

visualize non-specific cleavage patterns.
o Control Reactions: Include control reactions with known substrate DNA to assess the
enzyme's fidelity under specific conditions.
o Sequencing: Sequence the digested DNA fragments to confirm the precise cleavage sites
and identify any star activity.

Understanding and mitigating star activity are crucial for obtaining reliable and reproducible results
in molecular biology experiments that rely on the specificity of restriction enzymes, such as DNA
cloning and gene manipulation. Researchers should carefully optimize reaction conditions and
consider enzyme characteristics to minimize the risk of star activity.

Isoschizomers:

- Isoschizomers are different restriction enzymes that recognize the same or very similar DNA
sequences and cleave at identical or nearly identical positions within those sequences and
produce the same products.
- Despite being distinct enzymes, isoschizomers share specificity for a common or highly similar
DNA recognition site.
- This phenomenon allows researchers to substitute one isoschizomer for another in various
molecular biology applications, depending on factors such as enzyme availability, compatibility
with experimental conditions, or specific requirements for DNA manipulation.
- Example - EcoRI and PstI are classic examples of isoschizomers. While they are different enzymes
with different sources and specificities, they both recognize the sequence 5'-GAATTC-3'.

Neoschizomers:

- Isoschizomers that cleave at different sites within the same recognition sequence are called
Neoschizomers.
- They share specificity for the same recognition sequence but their cleavage sites are different,
ultimately producing different products.
- Example -

Frequency of Recognition Sequences:

If it’s assumed that the nucleotides are ordered in a random fashion and that the four different
nucleotides are present in equal proportions the restriction site of ‘n’ bp will occur on every ‘4n’
bp. For example, EcoRI, which recognizes a sequence of 6 bp will have a restriction site at every 4096
(46) bp. Thus, it’ll produce 4kb fragments.
Other Endonucleases:

• DNase I (Deoxyribonuclease I): DNase I is an endonuclease enzyme that cleaves internal

phosphodiester linkages within DNA molecules leading to the production of oligonucleotide
fragments.

o Key Features:
- Specificity: DNase I is relatively non-specific, cleaving both single-stranded and
double-stranded DNA. It hydrolyzes phosphodiester linkages, leading to the
generation of oligonucleotide fragments.
- Cofactor: Calcium ions (Ca²⁺) are required as a cofactor for the enzyme's activity. The
presence of Mg²⁺ can also influence its activity.
- Optimal Conditions: DNase I works optimally under slightly basic conditions (pH 7.5-
8.0).
- Heat Inactivation: The enzyme can be inactivated by heat treatment, typically at 65-
75°C for 10 minutes.
o Applications:
o RNA Purification: DNase I is used to remove contaminating genomic DNA from RNA
preparations. After RNA extraction, the sample is treated with DNase I to digest any
co-purified DNA.
o Chromatin Structure Studies: DNase I is employed to fragment chromatin in
chromatin accessibility assays. By treating isolated nuclei with DNase I, researchers
can study the accessibility of DNA to the enzyme, providing insights into chromatin
structure.
o Molecular Cloning: DNase I is used in certain molecular cloning procedures. For
example, in the creation of blunt-ended DNA fragments for cloning, DNase I can be
used to generate random breaks in the DNA.
o Mapping Protein-DNA Interactions: DNase I footprinting is a technique where the
enzyme is used to probe DNA-protein interactions. By digesting DNA with DNase I in
the presence of a DNA-binding protein, one can identify regions of protection
(footprints) where the protein is bound.

• S1 nuclease: S1 nuclease, is an endonuclease enzyme that specifically cleaves single-

stranded DNA (ssDNA) or RNA. The enzyme derived its name from its ability to act on single-
stranded regions of nucleic acids.

o Key Features:
- Substrate Specificity: S1 nuclease specifically cleaves single-stranded regions in DNA
or RNA molecules. It does not act on double-stranded regions.
- Mode of Action: S1 nuclease hydrolyzes phosphodiester bonds within the single-
stranded DNA or RNA, producing smaller fragments.
- Cofactors: The enzyme typically requires divalent cations, such as zinc ions (Zn²⁺), for
its activity.
- Optimal Conditions: S1 nuclease functions optimally under slightly acidic conditions
(pH 4.0-5.0).
 o Applications:
- Mapping Single-Stranded Regions: S1 nuclease is used to map single-stranded
regions in DNA or RNA. It is particularly valuable in identifying loops and other
structural features in nucleic acids.
- Hybridization Studies: S1 nuclease is used in hybridization studies to remove
unhybridized, single-stranded regions of DNA or RNA. This is useful for analyzing the
specificity and stability of nucleic acid hybridization.
- RNA Structure Analysis: In RNA structure studies, S1 nuclease can be used to assess
the secondary structure of RNA molecules by mapping single-stranded regions.
- Cloning and Recombinant DNA Technology: S1 nuclease can be employed in various
molecular cloning procedures. For example, it can be used to generate cohesive
ends in linearized plasmid DNA for subsequent cloning.
- Poly(A) Tailing: S1 nuclease is used in combination with other enzymes in poly(A)
tailing reactions to add polyadenine tails to the 3' ends of RNA molecules.

DIFFERENCES BETWEEN DNase 1 and S1 nuclease:

Characteristic DNase I S1 Nuclease

Type of Nuclease Endonuclease Endonuclease
Substrate Specificity Cleaves both single-stranded Preferentially cleaves single-
and double-stranded DNA stranded DNA
RNA Activity Generally, has minimal Cleaves single-stranded RNA
activity on RNA as well as DNA
Cofactors Generally requires divalent Requires divalent cations,
cations, such as Mg²⁺ or Ca²⁺ such as Zn²⁺
Optimal pH pH 7.5-8.0 pH 4.0-5.0
Heat Inactivation Can be inactivated by heat Can be inactivated by heat
treatment (typically 65-75°C) treatment (typically 65-75°C)
Applications Removal of DNA from RNA Mapping single-stranded
preparations, DNA regions in DNA/RNA, RNA
fragmentation, gene cloning structure analysis,
hybridization studies
Secondary Structure Does not significantly affect Can be used to assess the
DNA secondary structure secondary structure of RNA
Known Alternate Names None specifically associated Micrococcal nuclease,
with DNase I Staphylococcal nuclease
Source Organism Various sources, including Derived from Staphylococcus
bacteria and fungi aureus
DNA modifying enzymes and their applications:

1. DNA polymerases:
a. Their primary function is to catalyse the addition of deoxyribonucleotide triphosphates
to the growing DNA strand, facilitating the elongation of the DNA chain.
b. The synthesis of DNA happens in 5’ ⟶ 3’ direction.
c. Cause 3’ OH group is more electronegative than the 5’ OH group.
d. DNA polymerase is also involved in various DNA repair mechanisms such as base
excision repair, nucleotide excision repair, in mismatch repair. It helps in replacing or
excising damaged or incorrect nucleotides in the DNA sequence.

Different DNA polymerases used in Recombinant DNA technology:

• DNA polymerase I: (Kornberg polymerase) has both 3'⟶ 5’ and 5'⟶3’ exonuclease activity and
5'⟶3’ polymerase activity.
• Taq Polymerase: Taq polymerase is derived from the bacterium Thermus aquaticus and is widely
used in PCR. It is heat-stable, it lacks 3' to 5' exonuclease activity (proofreading ability), so it may
introduce errors during DNA synthesis. Optimum activity at ~ 72oC.
• Pfu Polymerase: Pfu (Pyrococcus furiosus) polymerase is another heat-stable DNA polymerase
used in PCR. Unlike Taq, Pfu has proofreading activity, resulting in higher fidelity during DNA
synthesis. This makes Pfu polymerase suitable for applications where accuracy is crucial.
• DNA Polymerase III: This is the primary enzyme involved in DNA replication in prokaryotes,
including E. coli. It is used in various cloning and amplification techniques, and some modified
versions with specific properties may be employed in recombinant DNA technology.

Applications of DNA polymerases:

• DNA replication.
• PCR.
• DNA sequencing.
• Site-directed mutagenesis - DNA polymerases are employed in site-directed mutagenesis to
introduce specific mutations into a DNA sequence. By using a modified DNA template and a DNA
polymerase that incorporates the desired mutations, researchers can generate specific changes
in the DNA sequence for functional studies.
• Cloning and gene expression.
• DNA repair.
• Next-Generation Sequencing.

2. Alkaline phosphatase:
• Removes phosphate groups from 5’ ends of DNA molecules which prevents these molecules
from being ligated to one another.
• Obtains energy from ATP hydrolysis.

Applications of Alkaline Phosphatase:

• Dephosphorylation of DNA.
• RNA labeling.
• ELISA.
• Western Blotting.
• Plasmid DNA Purification.

3. T4 Polynucleotide Kinase:
• T4 polynucleotide kinase obtained from E. coli cells infected with T4 phage performs the
reverse reaction to alkaline phosphatase, adding phosphates to 5’ ends.
• Catalyzes the transfer of the terminal (γ) phosphate from a nucleoside 5′ triphosphate to a 5′
hydroxyl group of a polynucleotide.
• Its main application is in the end labelling of DNA molecules.

Applications of T4 Polynucleotide Kinase:

• DNA and RNA labeling.

 • End repair and blunting.
• Linker ligation.
• DNA cloning.
• CDNA library construction.
• Site-Directed Mutation.
• DNA Footprinting.

4. Terminal deoxynucleotidyl transferase:

• It is a template independent DNA polymerase because it is able to synthesise a new DNA
polynucleotide without base pairing of the incoming nucleotides to an existing strand of
DNA or RNA.
• This enzyme is used for the formation of cohesive ends by homopolymer tailing. A
homopolymer is simply a polymer in which all the sub-units are the same.
• Enzyme terminal deoxynucleotidyl transferase catalyses the addition of a series of
nucleotides on to the 3’ hydroxyl terminal of double stranded DNA molecule. If this reaction
is carried out in the presence of just one type of deoxyribonucleotide, a homopolymer tail is
produced.

Applications of Terminal deoxynucleotidyl transferase:

• 3’ end labeling.
• Non-template nucleotide addition.
• Blunt-end cloning.
• Poly(dA) or poly(dT) tailing.
• RNA tailing.
• In Situ End Labeling (ISEL).
• DNA fingerprinting.
• Telomere length measurement.

DNA Ligases: DNA ligases join DNA molecules together by synthesizing phosphodiester bonds
between nucleotides at the ends of two different molecules, or at the two ends of a single molecule.
DNA ligases commonly used in cloning experiments are those obtained from E.coli or from the
bacteriophage T4.

Different DNA ligases:

1. T4 DNA Ligase: T4 DNA ligase is derived from the bacteriophage T4 and is widely used in
molecular biology and recombinant DNA technology. It catalyzes the ligation of cohesive
(sticky) or blunt-ended DNA fragments. T4 DNA ligase requires ATP as a cofactor for its
activity.
 2. T7 DNA Ligase: T7 DNA ligase is another ligase enzyme commonly used in molecular biology.
It is derived from the T7 bacteriophage and is particularly useful for ligating DNA fragments
with cohesive ends.
3. Pfu DNA Ligase: Pfu DNA ligase is derived from the archaeon Pyrococcus furiosus and is
known for its high fidelity in DNA ligation. It is often used when maintaining the accuracy of
the ligated DNA is crucial.
4. E. coli DNA Ligase: DNA ligase from Escherichia coli is also employed in various cloning and
recombinant DNA applications. It is often used for ligating DNA fragments during the
construction of recombinant plasmids.

Applications of DNA ligases:

• DNA cloning.
• DNA concatenation (joining multiple DNA fragments into longer sequences).
• Site-directed mutagenesis - DNA ligases are used in site-directed mutagenesis to ligate
mutated DNA fragments into a plasmid or other vectors.
• NGS library preparations.
• Real-time PCR probe ligation.
• Nick sealing in DNA replication.