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DNA digestion

Enzim-enzim yang menghidrolisis DNA :


1. Endonuklease : memotong DNA dari dalam
2. Eksonuklease : memotong DNA dari ujung
Restriction endonuclease
Restriction enzymes : DNAase that cleave DNA at specific site
Produced by bacteria
typically recognize the restriction sites (4- to 8-bp sequences)
cleave both DNA strands at this site.
commonly are short palindromic sequences the restriction-
site sequence is the same on each DNA strand when read in the 5 3
direction.

For each restriction enzyme, bacteria also produce a
modification enzyme, which protects a bacteriums own DNA
from cleavage by modifying it at or near each potential
cleavage site.

The modification enzyme adds a methyl group to one or two
bases, usually within the restriction site.
Restriction endonuclease
Restriction enzymes : DNAase that cleave DNA at specific site
When a methyl group is present there, the restriction
endonuclease is prevented from cutting the DNA.

Together with the restriction endonuclease, the methylating
enzyme forms a restriction-modification system that protects
the host DNA while it destroys incoming foreign DNA (e.g.,
bacteriophage DNA or DNA taken up during transformation) by cleaving it
at all the restriction sites in the DNA.

Many restriction enzymes make staggered cuts in the two DNA
strands at their recognition site, generating fragments that
have a single-stranded tail at both ends.
Restriction endonuclease
Restriction enzymes : DNAase that cleave DNA at specific site
The tails on the fragments generated at a given restriction site are
complementary to those on all other fragments generated by the
same restriction enzyme.

At room temperature, these single-stranded regions, often called
sticky ends, can transiently base-pair with those on other DNA
fragments generated with the same restriction enzyme.

A few restriction enzymes, such as AluI and SmaI, cleave both DNA
strands at the same point within the restriction site, generating
fragments with blunt (flush) ends in which all the nucleotides at
the fragment ends are base-paired to nucleotides in the
complementary strand.
Cleavage of DNA by the restriction enzyme EcoRI
This restriction enzyme from E. coli makes staggered cuts at the specific 6-bp inverted
repeat (palindromic) sequence shown, yielding fragments with single-stranded,
complementary sticky ends.

Many other restriction enzymes also produce fragments with sticky ends.
Restriction Enzyme

Source

DNA Sequence Recognized

Ends of Cleaved Molecule

EcoRI

Escherichia coli

5'G AATTC
3'CTTAA G
BamHI

Bacillus
amyloliquefaciens

5'G GATCC
3'CCTAG G
HindIII

Haemophilus influenzae

5'A AGCTT
3'TTCGA A

MstII

Microcoleus species

5'CCTNAGG
3'GGANTCC

TaqI

Thermus aquaticus

5'T CGA
3'AGCT
NotI

Nocardia otitidis

5'GCGGCCGC
3'CGCCGGCG
AluI*

Arthrobacter luteus

5'AGCT
3'TCGA
* = blunt ends






Restriction Enzyme: Source and Site of Action
Restriction Cleavage of DNA