ENZYMES USED UN GENETIC
ENGINEERING
There are many enzymes which are used in
genetic engineering as an important biological
tool. However, the important enzymes required
for genetic engineering are: restriction
endonucleases, DNA ligases, exonucleases,
DNA polymerases, alkaline phosphate,
polynucleotide kinase, terminal deoxynucleotidyl
transferase, reverse transcriptase, etc.
Restriction endonucleases are the special types
of endonucleases that cut DNA at speci c sites.
EXONUCLEASES
These enzymes act upon genome and digest the
base pairs on 5' or 3' ends of a single stranded
DNA or at single strand nicks or gaps in double
stranded DNA. On the basis of mode of action
the exonucleases are grouped into di erent
types:
Lambda exonuclease:
This enzyme removes nucleotides from 5’ end of
double stranded DNA. Consequently an
ff fi
, improved substrate for terminal transferase is
formed. Therefore, this enzyme is used for 5’ end
modi cation. The mode of action of this enzyme
is given below:
5’- GGAATT OH -3 —————>5’-GAATT OH-3
3’- CCTTAAp-5’. 3’-Cp-5’. + Np-5
Exonuclease III
This enzyme removes nucleotides from 3’-OH
end of the double stranded DNA fragment .
Consequently, as improved substrate is formed
which is used in gene manipulation. This enzyme
is used for 3’ end modi cation of DNA-fragment
as below:
5’- GGAATTOH-3’ 5’-GOH-3. +Np-5
3’- CCTTAAP-5’ ————>. 3’- CCTTAAP-5’
ENDONUCLEASES
They act upon genetic material and cleave the
double stranded DNA at any point except the
ends, but their actions involves only one strand
of the duplex.
fi fi
, RESTRICTION ENDONUCLEASES
The restriction enzymes are called as molecular
scissors . These act as foundation of
recombinant DNA technology. These enzymes
are present in bacteria and provide a type of
defence mechanism called the ‘restriction
modi cation system’. Molecular basis of these
systems was elucidated rst by Werner Arber in
1965. However, there was no understanding
about cutting and joining of DNA molecules
using DNA enzymes before 1970.
There are three di erent types of restriction
endonucleases: Type I, Type II and Type III. Each
enzyme di ers slightly by di erent mode of
action. Type I and Type Il are large multi subunit
complex which contains both endonuclease and
methylase activity. The di erent bacteria consist
of di erent restriction endonucleases, and in
correspondence to these the methylases too are
produced. These enzymes occur naturally in
bacteria as a chemical weapon against the
invading viruses and cut both strands of DNA
when certain foreign nucleotides are introduced
in the cell. These enzymes cleave a DNA to
ff
fi ff ff fi
ff ff
ENGINEERING
There are many enzymes which are used in
genetic engineering as an important biological
tool. However, the important enzymes required
for genetic engineering are: restriction
endonucleases, DNA ligases, exonucleases,
DNA polymerases, alkaline phosphate,
polynucleotide kinase, terminal deoxynucleotidyl
transferase, reverse transcriptase, etc.
Restriction endonucleases are the special types
of endonucleases that cut DNA at speci c sites.
EXONUCLEASES
These enzymes act upon genome and digest the
base pairs on 5' or 3' ends of a single stranded
DNA or at single strand nicks or gaps in double
stranded DNA. On the basis of mode of action
the exonucleases are grouped into di erent
types:
Lambda exonuclease:
This enzyme removes nucleotides from 5’ end of
double stranded DNA. Consequently an
ff fi
, improved substrate for terminal transferase is
formed. Therefore, this enzyme is used for 5’ end
modi cation. The mode of action of this enzyme
is given below:
5’- GGAATT OH -3 —————>5’-GAATT OH-3
3’- CCTTAAp-5’. 3’-Cp-5’. + Np-5
Exonuclease III
This enzyme removes nucleotides from 3’-OH
end of the double stranded DNA fragment .
Consequently, as improved substrate is formed
which is used in gene manipulation. This enzyme
is used for 3’ end modi cation of DNA-fragment
as below:
5’- GGAATTOH-3’ 5’-GOH-3. +Np-5
3’- CCTTAAP-5’ ————>. 3’- CCTTAAP-5’
ENDONUCLEASES
They act upon genetic material and cleave the
double stranded DNA at any point except the
ends, but their actions involves only one strand
of the duplex.
fi fi
, RESTRICTION ENDONUCLEASES
The restriction enzymes are called as molecular
scissors . These act as foundation of
recombinant DNA technology. These enzymes
are present in bacteria and provide a type of
defence mechanism called the ‘restriction
modi cation system’. Molecular basis of these
systems was elucidated rst by Werner Arber in
1965. However, there was no understanding
about cutting and joining of DNA molecules
using DNA enzymes before 1970.
There are three di erent types of restriction
endonucleases: Type I, Type II and Type III. Each
enzyme di ers slightly by di erent mode of
action. Type I and Type Il are large multi subunit
complex which contains both endonuclease and
methylase activity. The di erent bacteria consist
of di erent restriction endonucleases, and in
correspondence to these the methylases too are
produced. These enzymes occur naturally in
bacteria as a chemical weapon against the
invading viruses and cut both strands of DNA
when certain foreign nucleotides are introduced
in the cell. These enzymes cleave a DNA to
ff
fi ff ff fi
ff ff
