This document discusses various enzymes used for genetic engineering and DNA manipulation. It describes restriction endonucleases and DNA ligase which cut and join DNA fragments. It also discusses other DNA modifying enzymes like nucleases which degrade DNA, and polymerases which synthesize DNA copies. Specific enzymes covered in detail include DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, terminal transferase, T4 DNA ligase, and T4 RNA ligase.

1. NAME:- T.ARUNA
REG.NO:- U14BR001
BHARATH UNIVERSITY
DEPT OF GENETIC ENGINEERING
TECHNICAL SEMINOR

2. 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.

3. 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.

6. 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.

7. Nucleases and its action

8. 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.

9. 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)

10. 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

11. 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.

12. 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

13. 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.

14. 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.

15. 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

16. 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.

17. 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.

18. Ligases
 Fast and efficient ligation of DNA and RNA.
 T4 DNA Ligase
 T4 RNA Ligase

19. 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.

20. 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

21. 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

22. CONCLUSION:
 These are the modifying enzymes represent the
cutting and joining functions in DNA manipulation
and genetic engineering.

23. REFERENCES