1. Biotechnology and Recombinant DNA Chapter 9

2. I. Learning Objectives u Why genetic engineering? u Cloning basics u Beyond the basics

3. Overview of DNA manipulation

4. II. Why? u A. More efficient, less expensive production u B. Genetic information u C. Genetic alteration

5. III. Cloning ABC’s (Fig 9.1)

6. A. Requirements u 1. Target DNA u 2. Cloning vector u 3. Cut and paste enzymes u 4. Put DNA into host u 5. Selection procedure

7. 1. Target DNA u cDNA u Genomic DNA

8. 2. Vector choice u a. Why a vector? u b. Plasmids u c. Viral vectors u d. YACs, MACs and HACs

9. 3. Cut and paste enzymes (Fig 9.2) u Restriction enzymes u Ligase

11. 4. Put DNA into host cell u Transformation u Electroporation u Protoplast fusion u Microinjection

12. 5. Now, selection: Fig 9.11 u What is being selected? u Host grown on selective media

13. III. Beyond the basics u A. How do you find your gene of interest? u B. A most marvelous “work-around” cloning u C. How to “fingerprint” a killer

14. A. How do you find your gene of interest? u 1. Construct gene library u 2. Screen library

15. B. A marvelous “work-around” u 1. Polymerase Chain Reaction –Amplification of any target DNA u 2. Requires –target –DNA polymerase –primers u http://vector.cshl.org/Shockwave/pcranwho le.htm http://vector.cshl.org/Shockwave/pcranwho le.htm

16. 3. What’s it good for? u a. Identification of microbe in mixed culture –CD-ROM exercise u b. “Fingerprinting” a killer

17. b. Fingerprinting a killer u Tracking E. coli 0157:H7 (Fig 9.16)

18. Should we or shouldn’t we? u Safety issues u Ethical issues