Download Restriction Enzymes

Restriction Enzymes
• A Restriction Enzyme (or restriction
endonuclease) is an enzyme that cuts
double-stranded DNA at specific
recognition nucleotide sequences known
as restriction sites. Such enzymes, found
in bacteria and archaea, are thought to
have evolved to provide a defense
mechanism against invading viruses
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Restriction enzymes
• Restriction enzymes cut double-strand DNA at
specific recognition sequences which are often
4-6 base pair palindromes = 5’-3’ sequence is
identical on both DNA strands
• Many restriction enzymes cut the two DNA
strands at different points which generates
complementary single-strand ends = sticky ends
(others = blunt ends)
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Restriction Enzymes
• host DNA is methylated by a modification
enzyme (a methylase) to protect it from
the restriction enzyme’s activity
• Werner Arber discovered in 1962 enzyme
HindII (Haemophilus influenzae)
• the 1978 Nobel Prize for Physiology or
Medicine was awarded to Daniel Nathans,
Werner Arber, and Hamilton O. Smith
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2) Phương
pháp
(tt):
Digestion DNA
1. Cut DNA by restriction enzymes and
add enzyme ligase biding the complement
sequences
G
CTTAA
any double strand
TAA
T
EcoRI
MseI
GAATTC
CTTAA G
T TAA
AAT T
EcoRI
MseI
Any
double strand
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Restriction enzymes
BamHI (from B. amyloliquefaciens ) recognizes
GGATCC and cuts between the G’s on both strands
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Restriction enzymes
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Restriction enzymes
Restriction
enzymes
cut DNA
into defined
pieces,
named
restriction
fragments
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Restriction enzymes
DNA fragments of different size (e.g.
restriction fragments) can be separated
according to their size by gel electrophoresis:
• agarose gel electrophoresis (100 bp - 15 kb)
• polyacrylamide gel electrophoresis (1-500 b)
=PAGE
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