1. 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

2. 1. Restriction Endonucleases Restriction and modification systems in E. coli (W. Arber, 1960’s)

3. 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

4. Binding of the Restriction Enzyme BamHI to the DNA Helix

5. Recognition Sequences and Cutting Sites for some Restriction Endonucleases

6. Some Restriction Enzymes Used in Molecular Biology

7. Recognition Site of Restriction Enzyme EcoRI

8. Using Restriction Enzyme EcoRI to Produce Cohesive Ends

9. Types of Ends Generated by Different Restriction Enzymes

10. Generation of Recombinant DNA Using Restriction Enzymes

11. Restriction Mapping

12. Restriction Map

13. 2. DNA Modifying Enzymes a DNA Polymerase I 5’-3’ Exonuclease Activity 3’-5’ Exonuclease Activity DNA Polymerase Activity

14. The Klenow Fragment

15. Klenow fragment used for DNA sequencing

16. Reverse Transcriptase (RTase)

17. b nucleases

18. Deletion analysis using Bal 31 nuclease

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

20. ii Polynucleotide kinase

21. iii Alkaline Phosphatase: BAP and CIP

22. 3 DNA ligase

23. Mechanism of DNA ligase

24. DNA ligase used for both blunt and cohesive end ligation