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Understanding Genetic Engineering and Cloning in Biotechnology

This chapter explores the fundamentals of genetic engineering and recombinant DNA technology. It covers the importance of genetic alteration for efficient production and the intricacies of cloning, including necessary components such as target DNA, cloning vectors, and cut-and-paste enzymes. The chapter also delves into advanced techniques like gene libraries and polymerase chain reaction (PCR) for amplifying DNA. Additionally, ethical and safety issues associated with biotechnology practices are addressed, emphasizing the implications of manipulating genetic information.

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Understanding Genetic Engineering and Cloning in Biotechnology

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  1. Biotechnology and Recombinant DNA Chapter 9

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

  3. Overview of DNA manipulation

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

  5. III. Cloning ABC’s (Fig 9.1)

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

  7. cDNA Genomic DNA 1. Target DNA

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

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

  10. 4. Put DNA into host cell • Transformation • Electroporation • Protoplast fusion • Microinjection

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

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

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

  14. B. A marvelous “work-around” • 1. Polymerase Chain Reaction • Amplification of any target DNA • 2. Requires • target • DNA polymerase • primers • http://vector.cshl.org/Shockwave/pcranwhole.htm

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

  16. b. Fingerprinting a killer • Tracking E. coli 0157:H7 (Fig 9.16)

  17. Should we or shouldn’t we? • Safety issues • Ethical issues

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