Transcript Chapter 6: Analysis and Characterization of Nucleic Acids

Analysis and Characterization of
Restriction enzymes (RE)
1.
2.
Types of REs
Mapping of restriction enzyme sites
Restriction Enzymes
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Type I
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Type II
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Methylation/cleavage (3 subunits)
>1000 bp from binding site
e.g., Eco AI GAGNNNNNNNGTCA
Cleavage at specific recognition sites
Type III
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Methylation/cleavage (2 subunits)
24–26 bp from binding site
e.g., Hinf III CGAAT
Restriction Endonucleases: Type
II
Enzyme
Isolated
from
Recognition
sequence
Eco RI
E. coli, strain R,
Gν AATTC
1st enzyme
Eco RV
E. coli, strain R,
Gv ATATC
5th enzyme
Hind III
H. influenzae,
strain d,
3rd enzyme
Av AGCTT
Restriction Enzymes
Cohesive Ends
Cohesive Ends
Blunt Ends
(5´ Overhang)
(3´ Overhang)
(No Overhang)
BamH1
KpnI
HaeIII
GGATCC
CCTAGG
GGTACC
CCATGG
GGCC
CCGG
Restriction Enzymes
GGATCC
CCTAGG
AGATCT
TCTAGA
BamHI
BglII
BssSI
TspRI
(5’ Overhang)
(5’ Overhang)
(5’ Overhang)
(3’ Overhang)
CTCGTG NNCAGTGNN
GAGCAG NNGTCACNN
Enzymes Generating
Compatible Cohesive Ends
Enzymes Recognizing
Non palindromic Sequences
CCCGGG CCCGGG
GGGCCC GGGCCC
GATC
CTAG
GGCC
CCGG
DpnI
HaeIII
SmaI
XmaI
(Requires methylation)
(Inhibited by methylation)
(Blunt Ends)
(5’ Overhang)
Methylation-sensitive Enzymes
Isoschizomers
Ligation of Restriction Enzyme
Digested DNA
Sticky ends must match (be complementary)
for optimal re-ligation.
Blunt ends can be re-ligated with less efficiency
than sticky ends.
Sticky ends can be converted to blunt ends with
nuclease or polymerase. Blunt ends can be
converted to sticky ends by ligating to synthetic
adaptors.
Restriction Enzyme Mapping
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Digest DNA with a restriction enzyme.
Resolve the fragments by gel
electrophoresis.
The number of bands indicates the
number of restriction sites.
The size of the bands indicates the
distance between restriction sites.
Restriction Enzyme Mapping
BamH1 XhoI
BamH1
XhoI
BamH1
4.3 kb
3.7 kb
2.8 kb
4.0 kb
2.3 kb
1.9 kb
1.4 kb
1.3 kb
1.1 kb
2.8 kb
1.7 kb
1.7 kb
XhoI
1.2 kb 1.2 kb
1.2 kb
1.1 kb
0.7 kb
XhoI
Summary
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Restriction enzymes cut DNA at specific
recognition sequences.
DNA can be characterized by restriction
enzyme mapping.