ENZYMES INVOLVED IN GENE

MANIPULATION

Submitted to Dr Asif zahoor

Submitted by Fakiha javed
Roll # 15263
Bs microbiology
5th semester

Genetic manipulation:
It is also called genetic engineering.It involves
manualy adding new DNA to an organism to add
new traits.
Enzymes:
Enzyme are the proteins formed by cells and it is
used as a catalyst in many biological functions.
Enzymes involved in genome manipulation are
following:
Enzymes used in gene manipulation are
categorized into three classes. Such as:
Enzymes used in cutting, synthesis and joining.

Enzymes involved in joining:These

enzymes involved in the joining of DNA
such as ligase

Ligase:
It join DNA molecules together by
synthesizing phophodiaster bonds
between nucleotides at the ends of two
different molecules or at the two ends of a
signal molecule.

Source of ligase enzymes:

NA ligase from bacteriophage T4 it is mostly

used in laboratory research.it can ligate
sticky ends of DNA as well as RNA and DNA
hybrids.it uses ATP.
Sequence of DNA ligase:
It can proceed in either direction 5to3 or
3to5 depending on which end of the probe
oligonucleotides the 3to5 direction is more
efficient for doing multiple cycles of
ligation.it is opposite direction to
polymerase based sequencing methods.

Enzymes involved in
cutting:
Enzymes involved in cutting of DNA
like nucleases.

Nucleases:
Degradation of DNA is done by the
breaking of phophodiester bond that

link one nucleotide to the next.

Types of nucleases:
There are two types of nucleases
Exonuclease and endonuclease

Exonuclease:
These are the category of the
enzymes that cleave the nucleotides
at the end of DNA molecule e.g. T7
exonuclease isolated from
recombinant source purified from
E.Coli strain.snake venome and
spleen phosphodiasterase are the
examples of exonuclease.

Endonucleases:
These are the enzymes that
cause cleavage of bonds of DNA.

Action of endonuclease:
It attacks specific 3or5 linkages
in polypeptide chain. These are
divided into groupA&B.

Examples of
endonucleases:
Deoxyribonuclease I
&deoxyribose II.

Deoxyribonuclease I:
Source:
It is isolated from bovine
pancreas.

Deoxyribonuclease II:
It is another nuclease of class B.

Source:
It is isolated from spleen and
thymus of various bacteria.

Function:
It causes hydrolysis of 5
linkages.

Restriction enzymes
plays central role in all aspects of
recombinant DNA technology.

Action and sequence of

restriction
endonucleases:
It binds to a DNA molecule at a
specific sequence and makes a
double stranded cut at or near
that sequence.It cuts DNA in two
different ways.

Source:
Bacteria produce restriction
enzymes to fight against
bacteriophages.

Types of restriction
endonuclease:
There are three types of
restriction endonucleases :

Source of restriction
enzyme:
Bacteria produce restriction
enzymes to fight against
bacteriophages.

Types of restriction
endonuclease:
There are three types of
restriction endonucleases :

Type I enzyme:
It is a complex multisubunit
combination restriction and
modification enzyme that cut
DNA.it is capable of both
restriction and modification
activities.

Function of type I
enzyme:
It cuts DNA at random far from
there recognization sequences.

Sequence:
It cleave at random sites of about
thousand base pairs or more
from the recognization sequence
and it requires ATP for energy.

Type II:
It behaves slightly different and
does not require ATP.

Function:
It cut DNA at defined position
close to or with there recognition
sequences.They produce discrete
restriction fragments.
The most common type of type II
enzyme are those like
HhaI(NEB#R0139).

Sequence:
Most recognized DNA sequences
are symmetric because they bind
to DNA as homodimers but a few
recognize asymmetric DNA
sequence because r=they bind as
heterodimerS.

Type III:
These enzymes are also
combination of restriction and
modification enzymes. T

TypeIV:
It recognize modified typically
methylated DNA and are
exemplified by McrBC and Mrr
systems of E. Coli.

Sequence:
It acts as a heterodimer. Half
catalytic site per strand.it cuts
approximately 25basepairs from
recognition site also very large
with many subunits.

DNA modifying enzymes:

It involves many type of enzymes
such as alkaline phosphatase,
polynucleotide kinase, terminal
deoxynucleotidyle transferases
topoisomerases polymerases.

Alkaline phosphatase:
Source:
It is extracted from E.coli or calf
intestine.

Function:
This enzyme helps in modification
of DNA and helps in removal of
phosphate from many molecules
such as alkalides.

Sequence:
ECOcyc is a source of this
enzymes.

Polynucleotide kinase:
Source:
It is extracted from E.coli which is
infected with bacteriophages.

Example:
T4 polynucleotide kinase is
basically polynucleotide kinase.

Function:
It play role in modification of
DNA.It is an enzyme that catalyze
the transfer of phosphate from
ATP to 5 end of either DNA or
RNA.

Polymerases:
It synthesize the new strand of
DNA moleculecomplementry to
the existing DNA or RNA strand
which acts as template called as
template dependent DNA
polymerase .

Function:
Many of the techniques used to
study DNA depends on the
synthesis of copies of all or part
of existing DNA or RNA.

Source:
It can be extracted from E.coli.

Sequence:
DNA polymerase can add free
nucleotides only to the three end
of newly forming strand.

Transferase:
It is an enzyme which is
extracted from calf thymus
tissue which has got the capacity
to add one or more number of
nucleotide in 3 terminus of DNA
molecule.

Topoisomerases:
It changes the confirmation of
close circular DNA.

Function:
This enzyme helps in over winding or
under winding of DNA.