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

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 moleculesi 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