Biotechnology and Recombinant DNA

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Biotechnology and Recombinant DNA

• Chapter 9

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I. Learning Objectives

• Why genetic engineering?
• Cloning basics
• Beyond the basics

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Overview of DNA manipulation
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II. Why?

• A. More efficient, less expensive production
• B. Genetic information
• C. Genetic alteration

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III. Cloning ABCs (Fig 9.1)
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A. Requirements

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

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1. Target DNA

• cDNA

• Genomic DNA

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2. Vector choice

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

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3. Cut and paste enzymes (Fig 9.2)

• Restriction enzymes
• Ligase

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4. Put DNA into host cell

• Transformation
• Electroporation
• Protoplast fusion
• Microinjection

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5. Now, selection Fig 9.11

• What is being selected?
• Host grown on selective media

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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

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A. How do you find your gene of interest?

• 1. Construct gene library
• 2. Screen library

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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

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3. Whats it good for?

• a. Identification of microbe in mixed culture
• CD-ROM exercise
• b. Fingerprinting a killer

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b. Fingerprinting a killer

• Tracking E. coli 0157H7 (Fig 9.16)

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Should we or shouldnt we?

• Safety issues
• Ethical issues