Enzymes Used for Gene Engineering
1. Restriction endonuclease (restriction enzymes)
2. DNA modifying enzymes
a polymerases
b nucleases
c enzymes that modify the ends of DNA
molecules
3. DNA ligase
1. Restriction Endonucleases
Restriction and
modification
systems in E. coli
(W. Arber, 1960’s)
Three Types of Restriction Endonucleases
Type I: e.g. restriction enzymes in the
restriction-modification system
Type II: e.g. EcoRI, BamHI, HindIII
HindII (H. O. Smith, 1970)
Type III: e.g. MboII
Binding of the Restriction Enzyme BamHI to the DNA Helix
Recognition Sequences and Cutting Sites for some Restriction Endonucleases
Some Restriction Enzymes Used in Molecular Biology
Recognition Site of Restriction Enzyme EcoRI
Using Restriction Enzyme EcoRIto Produce Cohesive Ends
Types of Ends Generated by Different Restriction Enzymes
Generation of Recombinant DNA Using Restriction Enzymes
Restriction Mapping
2. DNA Modifying Enzymes a DNA Polymerase I
• 5’-3’ Exonuclease Activity
• 3’-5’ Exonuclease Activity
• DNA Polymerase Activity
The Klenow Fragment
Klenow fragment
used for DNA
sequencing
Reverse Transcriptase (RTase)
Deletion analysis using Bal 31 nuclease
c enzymes that modify the ends of DNA moleculesi Terminal transferase
ii Polynucleotide kinase
iii Alkaline
Phosphatase:
BAP and CIP
Mechanism of DNA ligase
DNA ligase used for both blunt and cohesive end ligation