GENETIC

ENGINEERING

Dr. Sidhartha Singh

Assistant Professor
DY Patil International University
Akurdi, Pune, India
 WHAT IS GENETIC ENGINEERING?
Genetic engineering is a field of biotechnology that involves manipulating the genetic material of organisms to introduce
specific traits or characteristics. It allows scientists to modify the DNA of an organism, including genes or gene sequences, in a
precise and controlled manner. This technology has wide-ranging applications in various fields, including agriculture, medicine,
industry, and environmental conservation

Conservation Recombinant
Biology DNA
technology

Genetically
CRISPR-
GE Modified
Cas9
Organisms

Biopharmace
Gene
utical
Therapy
Production

Cloning
 Recombinant Is a set of techniques that enable the DNA from different sources to be identified , isolated and
DNA recombined so that new characteristics can be introduced into an organism.
technology

1st recombinant DNA molecule that combined the DNA from SV40 virus and
Paul berg lambda phage

Essential property: the

Herbert W. Develop RDT technology by showing the genetically engineered DNA
desired DNA fragments
Boyer molecules may be cloned in foreign cells
must be selectively
amplified resulting in a
large increase in copy
y N. DNA Cloning
le number of selected DNA
t an hen sequences
S Co
 DNA Cloning Cell based

Cell Free

This was the 1st form of DNA cloning to be developed and is an invivo cloning
method. The 1st step is attaching foreign DNA fragments in vitro to DNA sequences,
capable of independent replication. The recombinant DNA fragments are then
transferred into suitable host cells where they can be propagated selectively. The
essence of cell based DNA cloning involves following steps:

1. Construction of recombinant DNA molecule: Recombinants are DNA molecules

consisting of autonomously replicating DNA segment plus inserted elements. Such
hybrid molecules are called as Chimera. Recombinant DNA molecules are
constructed by invitro covalent attachment (ligation) of the desired DNA fragments
(target DNA) to a replicon (any sequence capable of independent DNA replication).
This is step is facilitated by cutting the target DNA and replicon molecules with
specific restriction endonucleases before joining the different DNA fragments using
the enzyme DNA ligase
2. Transformation: The recombinant DNA molecules are transferred into host cells
(bacteria or yeast) in which chosen replication can undergo DNA replication
independently of the host cell chromosomes
3. Selective propagation of cell clones: it involves 2 stages: initially the transformed
cells are plated out by spreading on an agar surface in order to encourage the
growth of well separated cell colonies. These are cell clones. Subsequently,
individual colonies can be picked from the plate and the cells can be further
expanded in liquid cultures
4. Isolation of recombinant DNA clones: This can be done by harvesting expanded
cell cultures and selectively isolating the recombinant DNA

The polymerase chain reaction is a newer form of DNA cloning which is enzyme mediated and is conducted entirely in vitro. PCR, is techniques
used for selective amplification of specific target sequence of nucleic acid by using short primers.
 Enzymes for DNA manipulation
The enzymes used in RDT fall in to following 4 broad categories
After the staggered cuts, the
resulting restriction fragments
possess so-called 5′ overhangs
or 3′ overhangs. For example,
the recognition site for EcoRI
enzyme is 5′-GAATTC-3′. Once
the staggered cuts have been
made, the resulting fragments
have 5′ overhangs or
staggered ends. Similarly,
restriction enzyme PstI create
staggered cuts in the
recognition site (5′-CTGCAG-
3′) that results in 3′ overhangs
or staggered ends.
 Classification of Restriction Endonucleases:
• Type I restriction enzymes
• Type II restriction enzymes
• Type III restriction enzymes

Type I restriction enzymes: Type II restriction enzymes:

• These enzymes have both restriction and modification • Restriction and modification are mediated by separate
activities. Restriction depends upon the methylation status of the enzymes so it is possible to cleave DNA in the absence of
target DNA. modification. Although the two enzymes recognize the same
target sequence, they can be purified separately from each other.
• Cleavage occurs approximately 1000 bp away from the • Cleavage of nucleotide sequence occurs at the restriction site.
recognition site. • These enzymes are used to recognize rotationally symmetrical
sequence which is often referred as palindromic sequence.
• The recognition site is asymmetrical and is composed of two • These palindromic binding site may either be interrupted (e.g.
specific portions in which one portion contain 3–4 nucleotides BstEII recognizes the sequence 5´-GGTNACC-3´, where N can be
while another portion contain 4–5 nucleotides and both the parts any nucleotide) or continuous (e.g. KpnI recognizes the sequence
are separated by a non-specific spacer of about 6–8 nucleotides. 5´-GGTACC-3´).
• They require only Mg2+ as a cofactor and ATP is not needed for
• They require S-adenosylmethionine (SAM), ATP, and magnesium their activity.
ions (Mg2+) for activity. • Type II endonucleases are widely used for mapping and
reconstructing DNA in vitro because they recognize specific sites
• These enzymes are composed of mainly three subunits, a and cleave just at these sites .
specificity subunit that determines the DNA recognition site, a
restriction subunit, and a modification subunit
The steps involved in DNA binding and cleavage by a type II restriction
endonuclease:
• These enzymes have nonspecific contact with DNA and initially bind to DNA as dimmers.

• The target site is then located by a combination of linear diffusion or “sliding” of the
enzyme along the DNA over short distances, and hopping/jumping over longer distances.

• Once the target restriction site is located, the recognition process (coupling) triggers large
conformational changes of the enzyme and the DNA, which leads to activation of the
catalytic center

• Catalysis results in hydrolysis of phosphodiester bond and product release.

Structures of free, nonspecific, and specific DNA-bound forms of BamHI. The two dimers are shown in brown, the DNA backbone is in green and the
bases in gray. BamH I becomes progressively more closed around the DNA as it goes from the nonspecific to specific DNA binding mode.
Type III restriction enzymes:
• These enzymes recognize and methylate the same DNA sequence but cleave 24–26 bp away.
• They have two different subunits, in which one subunit (M) is responsible for recognition and modification of
DNA sequence and other subunit (R) has nuclease action.
• Mg+2 ions, ATP are needed for DNA cleavage and process of cleavage is stimulated by SAM.
• Cleave only one strand. Two recognition sites in opposite orientation are necessary to break the DNA duplex.
Type I and III restriction endonucleases are not useful for gene cloning because they cleave DNA at sites other
than the recognition sites and thus cause random cleavage patterns. In contrast, type II endonucleases are
widely used for cloning and mapping purposes because they recognize specific sites and cleave just at these
sites. Over 4000 type II restriction endonucleases have been isolated and characterized to date. Approximately
600 are available commercially for use by molecular biologists.
For the above problem the solution is modification of DNA ends
the 3'-OH terminus of the sticky end is kept unchanged, but the 5'
terminus is modified from 5'-P to 5'-OH terminus.
 Mechanism of Action of DNA Ligases

• ATP, or NAD+, reacts with the ligase enzyme to form a covalent enzyme–
AMP complex in which the AMP is linked to ε -amino group of a lysine
residue in the active site of the enzyme through a phosphoamide bond.

• The AMP moiety activates the phosphate group at the 5´-end of the
DNA molecule to be joined. It is called as the donor.

• The final step is a nucleophilic attack by the 3´-hydroxyl group on this

activated phosphorus atom which acts as the acceptor. A phosphodiester
bond is formed and AMP is released.

• The reaction is driven by the hydrolysis of the pyrophosphate released

during the formation of the enzyme–adenylate complex. Two high-energy
phosphate bonds are spent in forming a phosphodiester bond in the DNA
backbone with ATP serving as energy source.

• The temperature optimum for T4 DNA ligase mediated ligation in vitro is

16°C. However ligation is also achieved by incubation at 4°C by
incubating over night or at room temperature condition by incubating for
30 minutes.

• Adenylate and DNA-adenylate are the important intermediates of the

phosphodiester bond forming pathway.

The mechanism of DNA joining by DNA ligase.

 Deoxyribonuclease I (DNase I):
Deoxyribonuclease (DNase): An endonuclease which cleaves double-stranded DNA or single
stranded DNA. The cleavage preferentially occurs adjacent to
pyrimidine (C or T) residues. The major products are 5'-
• A nuclease enzyme that can catalyze the hydrolytic cleavage of phosphorylated bi-, tri- and tetranucleotides. It requires divalent ions
phosphodiester bonds in the DNA backbone are known as deoxyribonuclease (Ca2+ and Mn2+ /Mg2+) for its activity and creates blunt ends or 1-2
(DNase). overhang sequences.

• Based on the position of action, these enzymes are broadly classified as DNaseI is the most widely used enzyme in cloning experiments to
endodeoxyribonuclease (cleave DNA sequence internally) and remove DNA contamination from mRNA preparation (to be used for
exodeoxyribonuclease (cleave the terminal nucleotides). cDNA library preparation, northern hybridization, RT-PCR etc). The
mode of action of DNaseI varies according to the divalent cation used.
• Unlike restriction enzymes, DNase does not have any specific
recognition/restriction site and cleave DNA sequence at random locations. In the presence of magnesium ions (Mg+2), DNaseI hydrolyzes each
strand of duplex DNA producing single stranded nicks in the DNA
• There is a wide variety of deoxyribonucleases known which have different backbone, generating various random cleavages.
substrate specificities, chemical mechanisms, and biological functions. They
are: On the other hand, in the presence of manganese ions (Mn +2), DNaseI
cleaves both strands of a double stranded DNA at approximately the
same site, producing blunt ended DNA fragments or with 1-2 base
overhangs. The two major DNases found in metazoans are:
deoxyribonuclease I and deoxyribonuclease II.

Some of the common applications of DNase I in rDNA technology have

been mentioned below:
• Eliminating DNA contamination (e.g. plasmid) from preparations of
RNA.
• Analyzing the DNA-protein interactions via DNA footprinting.
• Nicking DNA prior to radio-labeling by nick translation.

Action of DNase I in the presence of Mg+2 and Mn+2 ions. (Arrowhead

denoting random site of cleavage in double stranded DNA by DNase I)
DeoxyribonucleaseII (DNaseII):

It is a non-specific endonuclease with optimal activity at acidic pH (4.5-5.5) and

conserved from human to [Link]. It does not require any divalent cation for its
activity. DNaseII initially introduces multiple single stranded nicks in DNA backbone
and finally generates 3' phosphate groups by hydrolyzing phosphodiester linkages.

This enzyme releases 3' phosphate groups by hydrolyzing phosphodiester linkage

and creating nicks in the DNA backbone. DNaseII acts by generating multiple single
stranded nicks followed by production of acid soluble nucleotides and
oligonucleotides. The catalytic site of the enzyme contains three histidine residues
which are essential for enzyme activity.
Exonuclease III:
Some of the common applications of DNase II are as follows:
• DNA fragmentation Exonuclease III is a globular enzyme which has 3'→5' exonuclease
• Molecular weight marker activity in a double stranded DNA. The template DNA should be
• Cell apoptosis assays etc. double stranded and the enzyme does not cleave single stranded
DNA. The enzyme shows optimal activity with blunt ended sequences
or sequences with 5' overhang.
Mung bean nuclease:
As the name suggest, this nuclease enzyme is isolated from mung bean Exonuclease III enzyme has a bound divalent cation which is essential
sprouts (Vigna radiata). Mung bean nuclease enzymes can degrade single for enzyme activity. The mechanism of the enzyme can be affected
stranded DNA as well RNA. Under high enzyme concentration, they can by variation in temperature, monovalent ion concentration in the
degrade double stranded DNA, RNA or even DNA/RNA hybrids. reaction buffer, and structure and concentration of 3' termini. The
enzyme shows optimal activity at 37°C at pH 8.0.
Mung bean nuclease can cleave single stranded DNA or RNA to produce 5'-
phosphoryl mono and oligonucleotides. It requires Zn 2+ ion for its activity and Various application of exonuclease III in molecular cloning
shows optimal activity at 37°C. The enzyme works in low salt concentration experiments are:
(25mM ammonium acetate) and acidic pH (pH 5.0). Treatment with EDTA or • To generate template for DNA sequencing
SDS results in irreversible inactivation of the enzyme. • To generate substrate for DNA labeling experiments
• Directed mutagenesis
Mung bean nuclease is less robust than S1 nuclease and easier to handle. It • DNA-protein interaction assays (to find blockage of exonuclease III
has been used to create blunt end DNA by cleaving protruding ends from 5' activity by protein-DNA binding) etc.
ends. This enzyme cannot produce nicks in a double stranded DNA but at
higher concentration, it can generate nicks and cleave double stranded DNA.
DNA polymerase:
• DNA polymerase is a complex enzyme which synthesize
nucleotide complementary to template strand.
• It adds nucleotide to free 3′ OH end and help in elongation of
strand
• It also helps to fill gap in double stranded DNA.
• DNA polymerase-I isolated from E. coli is commonly used in
gene cloning
• Taq polymerase isolated from Thermus aquaticus is used in
PCR
Ribonuclease-H (RNase H):
• RNase-H removes mRNA from DNA-RNA heteroduplex and that
mRNA is used to synthesize cDNA
• It is isolated from retrovirus