NAME:- T.

ARUNA
REG.NO:- U14BR001
BHARATH UNIVERSITY
DEPT OF GENETIC ENGINEERING
TECHNICAL SEMINOR
 Such manipulations of DNA are
conducted by a toolkit of enzymes:
 restriction endonucleases are used as molecular scissors,
 DNA ligase functions to bond pieces of DNA together, and

 a variety of additional enzymes that modify DNA are used to

facilitate the process.
DNA modifying enzymes
 Restriction enzymes and DNA ligases represent the
cutting and joining functions in DNA manipulation.
 All other enzymes involved in genetic engineering fall
under the broad category of enzymes known as DNA
modifying enzymes.
 These enzymes are involved in the degradation,
synthesis and alteration of the nucleic acids.
Nucleases
 Nuclease enzymes degrade nucleic acids by breaking
the phosphodiester bond that holds the nucleotides
together.
 Restriction enzymes are good examples of
endonucleases, which cut within a DNA strand.
 A second group of nucleases, which degrade DNA
from the termini of the molecule, are known as
exonucleases.
Nucleases and its action
Polymerases
 Polymerase enzymes synthesise copies of nucleic acid
molecules and are used in many genetic engineering
procedures.
 When describing a polymerase enzyme, the terms ‘DNA-
dependent’ or ‘RNA-dependent’ may be used to indicate
the type of nucleic acid template that the enzyme uses.
 Thus, a
 DNA-dependent DNA polymerase copies DNA into DNA,
 an RNA-dependent DNA polymerase copies RNA into DNA,
and
 a DNA-dependent RNA polymerase transcribes DNA into
RNA.
DNA Polymerases
 Mesophilic and thermophilic DNA
polymerases for different polymerization
reactions, DNA end blunting and
amplification, labeling and others.
 DNA Polymerase, Large Fragment
 DNA Polymerase I
 T4 DNA Polymerase
 T7 DNA Polymerase
 Terminal Transferase (TdT)
DNA Polymerase, Large Fragment
•DNA Polymerase, Large Fragment, is a portion of
DNA polymerase of Bacillus smithii, which catalyzes
5'=>3' synthesis of DNA and lacks 5'→3' and 3'→5'
exonuclease activities.

Highlights
 Thermophilic DNA polymerase with strong strand
displacement activity
DNA Polymerase I
 DNA Polymerase I, a template-dependent DNA
polymerase, catalyzes 5'→3' synthesis of DNA.
 The enzyme also exhibits 3'→5' exonuclease
(proofreading) activity, 5'→3' exonuclease activity, and
ribonuclease H activity.
Highlights
Incorporates modified nucleotides
Active in multiple buffers, including restriction
enzyme, PCR, and RT buffers

Applications
 DNA labeling
 Second-strand synthesis of cDNA in
conjunction with RNaseH
T4 DNA Polymerase
 T4 DNA Polymerase, a template-dependent DNA
polymerase, catalyzes 5'-3' synthesis from primed
single-stranded DNA.
 The enzyme has a 3'-5' exonuclease activity, but lacks
5'-3' exonuclease activity.
T7 DNA Polymerase
 T7 DNA Polymerase, a template dependent DNA
polymerase.
 It catalyzes DNA synthesis in the 5'=>3' direction.
 It is a highly processive DNA polymerase allowing
continuous synthesis of long stretches of DNA.
Applications

 Purification of covalently closed circular DNA by

removal of residual genomic DNA
 Primer extension reactions on long templates
 DNA 3'-end labeling
 Strand extensions in site-directed mutagenesis
Terminal Transferase (TdT)
 Protruding, recessed or blunt ended double or single
stranded DNA molecules serve as a substrate for TdT.

 TdT is isolated and purified from an E. coli strain

carrying the cloned terminal transferase gene from calf
thymus.
 DNA ligase
 DNA ligase is an important cellular enzyme, as its
function is to repair broken phosphodiester bonds that
may occur at random or as a consequence of DNA
replication or recombination.

 It can therefore be thought of as molecular glue, which

is used to stick pieces of DNA together.
Ligases
 Fast and efficient ligation of DNA and RNA.
 T4 DNA Ligase
 T4 RNA Ligase
T4 DNA Ligase
 The enzyme repairs single-strand nicks in duplex
DNA, RNA, or DNA/RNA hybrids.

 It also joins DNA fragments with either cohesive or

blunt termini, but has no activity on single-stranded
nucleic acids.

 The T4 DNA Ligase requires ATP as a cofactor.

Applications

 Cloning of restriction enzyme generated DNA

fragments
 Cloning of PCR products
 Joining of double-stranded oligonucleotide
linkers or adaptors to DNA
 Site-directed mutagenesis
T4 RNA Ligase
 T4 RNA Ligase catalyzes the ATP-dependent intra-
and intermolecular formation of phosphodiester
bonds between 5'-phosphate and 3'-hydroxyl termini
of oligonucleotides, single-stranded RNA and DNA.

Applications
 Joining RNA to RNA
 Specific modifications of tRNAs
 Site-specific generation of composite primers for PCR
CONCLUSION:
 These are the modifying enzymes represent the
cutting and joining functions in DNA manipulation
and genetic engineering.
REFERENCES