Enzymes and recombinant DNA technology

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ENZYMES AND RECOMBINANT
DNA TECHNOLOGY
Introduction
A recombinant DNA (Deoxyribose Nucleic Acid) molecule
is a vector (for example, a plasmid, phage or virus) into
which the desired DNA fragment has been inserted to enable
its cloning in an appropriate host. This is achieved by using
specific enzymes for cutting the DNA (restriction enzymes)
into suitable fragments and then for joining or ligating
appropriate fragments (ligase enzyme).
All these steps concerned with piecing together DNA
segments of diverse origin and then placing them into a
suitable vector constitute Recombinant DNA Technology;
popularly known as Genetic Engineering.
Recombinant DNA Technology
Recombinant DNA is formed by combining DNA from two
different organisms. It can be referred to as creation of a
new combination of DNA segments that are not found in
nature.
Recombinant DNA technology involves the following
sequential steps:-
1. DNA fragments coding for gene of interest are
synthesized chemically or isolated from an organism.
This can be achieved by treating bacterial cell/ plant or
animal tissue with enzymes such as lysozyme (bacteria),

2. cellulase (plant cell), chitinase (fungus), ribonuclease
(RNA) and protease (
2. Cutting of DNA fragment (region with gene of interest)
and vector DNA at specific locations using restriction
enzymes. Restriction enzyme digestions are performed
by incubating purified DNA molecules with the
restriction enzyme at the optimal co
specific enzyme.
3. The joining of required DNA fragment with vector DNA
using ligase enzyme. For this, the cut out gene of interest
from the source DNA and the cut vector are mixed and
ligase is added. This results in preparation of
recombinant DNA.
4. The recombinant DNA molecules are
into a host to replicate.
5. Recipient host cells that have acquired the recombinant
DNA are selected. The desired clones are then
characterised to ensure that they maintain true copies of
DNA segment that
Figure
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cellulase (plant cell), chitinase (fungus), ribonuclease
(RNA) and protease (protein).
Cutting of DNA fragment (region with gene of interest)
and vector DNA at specific locations using restriction
. Restriction enzyme digestions are performed
by incubating purified DNA molecules with the
restriction enzyme at the optimal conditions for that
The joining of required DNA fragment with vector DNA
using ligase enzyme. For this, the cut out gene of interest
from the source DNA and the cut vector are mixed and
ligase is added. This results in preparation of
nant DNA.
The recombinant DNA molecules are now introduced
into a host to replicate.
Recipient host cells that have acquired the recombinant
DNA are selected. The desired clones are then
characterised to ensure that they maintain true copies of
that was originally cloned.
Figure 1. Recombinant DNA Technology
cellulase (plant cell), chitinase (fungus), ribonuclease
Cutting of DNA fragment (region with gene of interest)
and vector DNA at specific locations using restriction
. Restriction enzyme digestions are performed
by incubating purified DNA molecules with the
nditions for that
The joining of required DNA fragment with vector DNA
using ligase enzyme. For this, the cut out gene of interest
from the source DNA and the cut vector are mixed and
ligase is added. This results in preparation of
now introduced
Recipient host cells that have acquired the recombinant
DNA are selected. The desired clones are then
characterised to ensure that they maintain true copies of

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Such an isolation and manipulation of genes allows more
precise genetic analysis and also has practical applications
in medicine, agriculture and industries.
Tools of Recombinant DNA Technology
Recombinant technology utilizes certain biological products
and biological agents for achieving its objectives; these are
called genetic engineering tools or tools for recombinant
DNA technology. The basic tools are:-
1. Restriction endonuclease enzyme – To cut or cleave the
DNA at specific sites.
2. Vector – A suitable DNA molecule capable of self
replication in a selected host cell. The DNA fragment to
be cloned is integrated into the vector. For example,
plasmid pBR322.
3. DNA ligase enzyme – To join the required fragment of
DNA with the vector.
4. Host – A suitable organism for the propagation of the
recombinant DNA, i. e., DNA insert.
Enzymes used in Recombinant DNA
Technology
The enzymes act as an important tool in genetic engineering.
The important ones are discussed below:
1. RESTRICTION ENDONUCLEASE
Restriction enzymes are ‘molecular scissors’ involved in
the cutting or fragmentation of DNA molecules. These
enzymes are a part of a DNA immunity system in

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bacteria, protecting the cell against entry of foreign DNA
by catalyzing double strand cleavages.
Endonuclease is a type of nuclease enzyme, which
degrade nucleic acids by cleaving phosphodiester
linkages. Thus endonuclease type of restriction enzyme
recognize specific sequences in the DNA and break the
DNA chain at those points; which are referred as
recognition sequence.
The restriction endonuclease recognizes a specific
palindromic nucleotide sequences in the DNA. The
palindrome in DNA is a sequence of base that reads same
on the two strands when oriention of reading is kept the
same. For example, the following sequences read the
same on the two strands in 5’ → 3’ direction. This is also
true if read in the 3’ → 5’ direction.
5’ ― GAATTC ― 3’
3’ ― CTTAAG ― 5’
 Some restriction enzymes cut the strand of DNA a
little away from the centre of the palindrome sites, but
between same two bases on the opposite strand. This
leaves single stranded portions at the ends. These are
overhanging stretches called sticky ends, with exposed
nucleotides, on each strand. For example, EcoRI cuts
in the following manner to produce Sticky ends.

5.  Other restriction enzymes cut the
from the centre of the palindromic sequence
generating flush ends with no exposed nucleotides.
These are called Blunt ends. For example, HindII cuts
in the following manner to produce blunt ends.
Restriction endonucleases are an indispensible part of
recombinant DNA technology. Both, the vector and the
required gene fragment, are cut using same restriction
endonuclease thus producing complimentary sticky ends.
2. DNA LIGASE
DNA ligase is the glue
the ends of DNA together.
the DNA fragments are joined by these enzymes to form
recombinant DNA.
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Figure 2. Sticky Ends
Other restriction enzymes cut the strands of DNA
from the centre of the palindromic sequence
generating flush ends with no exposed nucleotides.
These are called Blunt ends. For example, HindII cuts
in the following manner to produce blunt ends.
Figure 3. Blunt Ends
Restriction endonucleases are an indispensible part of
recombinant DNA technology. Both, the vector and the
required gene fragment, are cut using same restriction
endonuclease thus producing complimentary sticky ends.
LIGASE
is the glue of molecular genetics that holds
the ends of DNA together. The sticky ends so formed in
the DNA fragments are joined by these enzymes to form
recombinant DNA.
strands of DNA
from the centre of the palindromic sequence
generating flush ends with no exposed nucleotides.
These are called Blunt ends. For example, HindII cuts
in the following manner to produce blunt ends.
Restriction endonucleases are an indispensible part of
recombinant DNA technology. Both, the vector and the
required gene fragment, are cut using same restriction
endonuclease thus producing complimentary sticky ends.
of molecular genetics that holds
The sticky ends so formed in
the DNA fragments are joined by these enzymes to form

6. All living cells produce ligase that joins together the
neighbouring nucleotides to seal discont
DNA strands. In vitro, DNA ligase joins together two
DNA molecules during the production of recombinant
DNA. Usually, it is prepared from E. coli cells infected
with phage T4 and thus the most commonly used ligase is
T4 DNA ligase.
The DNA fragments are joined together
pairing rule, i.e.,
hydrogen bonds and Cytosine (C) joins to Guanine (G)
by three hydrogen bonds. The exposed nucleotides of the
fragments of DNA are linked by catalytic act
enzyme DNA ligase which joins the fragments at their
sugar phosphate backbone by creating a phosphodiester
bond between the two ends.
Thus DNA ligase is
of a recombinant
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All living cells produce ligase that joins together the
neighbouring nucleotides to seal discontinuity in the
DNA strands. In vitro, DNA ligase joins together two
DNA molecules during the production of recombinant
DNA. Usually, it is prepared from E. coli cells infected
with phage T4 and thus the most commonly used ligase is
agments are joined together using the base
Adenine (A) joins to thymine (T) by two
hydrogen bonds and Cytosine (C) joins to Guanine (G)
by three hydrogen bonds. The exposed nucleotides of the
fragments of DNA are linked by catalytic act
enzyme DNA ligase which joins the fragments at their
sugar phosphate backbone by creating a phosphodiester
bond between the two ends.
Figure 4. DNA Ligation
Thus DNA ligase is an essential enzyme in the formation
of a recombinant DNA molecule.
All living cells produce ligase that joins together the
inuity in the
DNA strands. In vitro, DNA ligase joins together two
DNA molecules during the production of recombinant
DNA. Usually, it is prepared from E. coli cells infected
with phage T4 and thus the most commonly used ligase is
using the base
Adenine (A) joins to thymine (T) by two
hydrogen bonds and Cytosine (C) joins to Guanine (G)
by three hydrogen bonds. The exposed nucleotides of the
fragments of DNA are linked by catalytic action of
enzyme DNA ligase which joins the fragments at their
sugar phosphate backbone by creating a phosphodiester
the formation

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References:-
1. Enzyme Structure and Mechanism; Alan Fersht
2. Modern’s abc of Biology; B.B. Arora & A.K. Sabharwal
3. Biotechnology Expanding Horizons; B. D. Singh
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