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 EcoRI to Produce Cohesive Ends

Types of Ends Generated by Different Restriction Enzymes

Generation of Recombinant DNA Using Restriction Enzymes

Restriction Mapping

Restriction Map

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)

b nucleases

Deletion analysis using Bal 31 nuclease

c enzymes that modify the ends of DNA molecules i Terminal transferase

ii Polynucleotide kinase

iii Alkaline Phosphatase: BAP and CIP

3 DNA ligase

Mechanism of DNA ligase

DNA ligase used for both blunt and cohesive end ligation