1. Analysis and Characterization of Restriction enzymes (RE)
Types of REs
Mapping of restriction enzyme sites

2. Restriction Enzymes Type I Type II Type III
Methylation/cleavage (3 subunits)
>1000 bp from binding site
e.g., Eco AI GAGNNNNNNNGTCA
Type II
Cleavage at specific recognition sites
Type III
Methylation/cleavage (2 subunits)
24–26 bp from binding site
e.g., Hinf III CGAAT

3. Restriction Endonucleases: Type II
Enzyme
Isolated from
Recognition sequence
Eco RI
E. coli, strain R,
1st enzyme
Gν AATTC
Eco RV
5th enzyme
Gv ATATC
Hind III
H. influenzae, strain d,
3rd enzyme
Av AGCTT

5. Restriction Enzymes BamH1 HaeIII KpnI Cohesive Ends Blunt Ends GGATCC
CCTAGG
HaeIII
GGCC
CCGG
Cohesive Ends
(5´ Overhang)
(3´ Overhang)
KpnI
GGTACC
CCATGG
Blunt Ends
(No Overhang)

6. Restriction Enzymes GATC CTAG GGCC CCGG CCCGGG GGGCCC GGATCC CCTAGG
DpnI
(Requires methylation)
Methylation-sensitive Enzymes
GGCC
CCGG
HaeIII
(Inhibited by methylation)
CCCGGG
GGGCCC
XmaI
(5’ Overhang)
SmaI
(Blunt Ends)
Isoschizomers
Enzymes Generating
Compatible Cohesive Ends
GGATCC
CCTAGG
BamHI
AGATCT
TCTAGA
BglII
CTCGTG
GAGCAG
BssSI
NNCAGTGNN
NNGTCACNN
TspRI
(3’ Overhang)
Enzymes Recognizing
Non palindromic Sequences

7. Ligation of Restriction Enzyme Digested DNA
Sticky ends must match (be complementary)
for optimal re-ligation.
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.
Blunt ends can be re-ligated with less efficiency than sticky ends.

8. Restriction Enzyme Mapping
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.

9. Restriction Enzyme Mapping
4.3 kb
3.7 kb
2.3 kb
1.9 kb
1.4 kb
1.3 kb
0.7 kb
BamH1
XhoI
4.0 kb
2.8 kb
1.2 kb
1.7 kb
1.1 kb
BamH1
XhoI
1.1 kb
1.7 kb
1.2 kb
2.8 kb

10. Summary Restriction enzymes cut DNA at specific recognition sequences.
DNA can be characterized by restriction enzyme mapping.