The slides on the DNA modifying enzymes

1. DNA MODIFYING ENZYMES
PRESENTED BY
DASTHAGIRI PASHA S

2. 1. DNA modifying enzymes
2. Polymerase
3. DNase
4. RNase
5. Polynucleotide kinase
6. Alkaline phosphatase
7. Nucleases
CONTENT

6. APPLICATIONS

7. DNA modifying enzymes --
❖ DNA modifying enzymes are involved in
genetic engineering.
❖ Restriction enzymes and DNA ligase
represent the cutting and joining
function in DNA manipulation.
❖ These enzymes are involved in the
degradation, synthesis and alternation of
the nucleic acid

8. POLYMERASES
❖ DNA polymerase are enzymes that synthsis a new
strand of DNA complementary to an existing DNA or
RNA template .
❖ Polymerases can function only if the template
possess a double strandedregion that act as a
primer for initiation of polymerization.
❖ The polymerase and nuclease activities of DNA
polymerase 1 are controlled by different parts of
enzyme molecule.
❖ These enzymes attaches to a short single - stranded
region in a mainly double stranded DNA molecule ,
and then synthesis a completely new strand ,
degrading the existing strand as it proceed

9. The polymerase can be
classified into 3 types they are
a) DNA dependentDNA polymerase that
copies DNA from DNA .
b)RNA dependentRNA polymerase that
synthesis DNA from RNA
c) DNA dependant RNA polymerase that
produces RNA from DNA.

10. DEOXY RIBONUCLEASE
❏ A nucleaseenzymethat can catalyse the hydrolytic cleavage
of phosphodiesterbond in the DNA backbone are known as
Deoxy ribonuclease.
❏ These enzymes are broadly classified as endo and exo
deoxyribonuclease
❏ DNase does not have specieficrecognition site and cleave
DNA sequenceat random location
❏ DNase are wide variety which known different substrate
specificities,chemicalmechanism , and biological functions.

11. ➢ They are two types of DNase -
➢ DNase1
➢ DNase2
○ 1.DNase1 :-
○ They which cleavedouble strandedDNA or single
strandedDNA.
○ The major products are 5’- phosphorylated,bi,tri, and
tetranucleotides.
○ The presence of manganeseions ,DNase1 hydrolyze each
strand of duplex DNA producingsingle strandedNick's
in the DNA backbone,generating variousrandom
cleavages.

12. Applications of DNase 1
➢Eliminating DNA contamination
from preparation of RNA.
➢Analyzing the DNA -protien
interaction via DNA foot printing.
➢Nicking DNA prior to radio labeling
by Nick translation.

13. DNase 2 :-
● It's a non speciefic endonucleases
pH is (4.5-5.5)
● Dnase 2 initially introduce multiple
single stranded Nick's in DNA
backbone .
● Generating single stranded Nick's by
producing of acid soluble nucleotide
and oligonucleotide .

14. Applications of DNase 2 :-
❖ DNA fragmentation
❖ Molecular weight marker
❖ Cell apoptosis assays etc...

15. Ribonuclease (RNase )

16. ● Nuclease that can catalyse hydrolysis of
ribonucleotide from either single
stranded or double stranded RNA
sequence are called ribonucleotide .
● RNA is important for RNA maturation
and processing .
● RNase A and RNase H play important
role in initial defence mechanism against
RNA viral infection.

17. Applications of RNase :-
● Eliminating or reducing RNA
contamination in preparation of plasmid
DNA.
● Mapping mutation in DNA or RNA by
mismatch cleavage .RNase will cleave
the RNA in RNA-DNA hybrid at sites of
single base mismatch & the cleavage
product can be analysed.
● Useful of RNA sequencing.

18. POLYNUCLEOTIDE KINASE
➔ PNK is a tetramer with phosphatase
activity at 3’ end and kinase at 5’ end
with tunnellike active site.
➔ PNK catelysed the transfer of a p04
group from gamma position of ATP to
the 5’end of either DNA or RNA .
➔ Basic residue of active site of PNK
8nteract with -vely charged phosphate
of the DNA .

21. APPLICATIONS OF PNK
❏ The linkers and adapter are
phosphorylated along with the
fragment of DNA before
ligation,which requires a 5’
phophate .this include products of
PCR ,which are generated by using
non phosphorylated primer
❏ PNK is also used for radiolabelling
oligonucleotides

22. ALKALINE PHOSPHATASE
● It is homodimer enzyme
● Optal pH is about 10
● Alkaline phosphate is closed to metal
ions that is mg & zn .
● Human body has present 4 isoforms
● The genes encode tissue specific
isoform are present on chromosome -2

23. They are 3 Alkaline phosphate are
used in gene manipulation
❖ Bacterial alkaline phosphate
(BAP)
❖ Calf intestinal alkaline
phosphate (CIP)
❖ Shrimp alkaline phophate (SAP)

24. APPLICATIONS OF ALKALINE
PHOSPHATASE

25. NUCLEASES

26. APPLICATIONS OF NUCLEASES

27. CONCLUSION

28. References
➢ Paul A .genetic engineering.in.genetic from gene to
genome
➢ Garden j e principle of genetics wily India pvt.ltd
new delhi
➢ Old R W and primrose H B (1980) principle og gene
manipulation.
➢ http//www.onlinebiologynotes.com