Biotechnology and Recombinant DNA

1. Biotechnology and Recombinant DNA • Biotechnology: • The use of microorganisms, cells, or cell components to make a product • Foods, antibiotics, vitamins, enzymes • Recombinant DNA Technology: • Insertion or modification of genes to produce desired proteins

2. DNA Cloning Figure 9.1.1

3. DNA Cloning Figure 9.1.2

4. E. coli • Used because it is easily grown and its genomics are known • Need to eliminate endotoxin from products • Cells must be lysed to get product

5. Recombinant Therapeutics Table 9.1.1

6. Recombinant Therapeutics Table 9.1.2

7. Selection & Mutation • Selection: Culture a naturally-occurring microbe that produces desired product • Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait • Site-directed mutagenesis: Change a specific DNA code to change a protein

8. Restriction Enzymes • Used in Cloning • Cut specific sequences of DNA • Isolated from bacterial cells

9. Use of Restriction Enzymes Figure 9.2

10. Vectors • Carry new DNA to desired cell • Shuttle vectors can exist in several different species • Plasmids and viruses can be used as vectors Figure 9.3

11. Polymerase Chain Reaction (PCR) • To make multiple copies of a piece of DNA enzymatically • Used to • Clone DNA for recombination • Add restriction sites to your gene • Sequence DNA • Diagnose genetic disease • Detect pathogens

12. PCR Figure 9.4.1

13. PCR Figure 9.4.2

14. How Do Scientists Insert DNA into a Cells? • Transformation • Transduction (Ch. 12) • Electroporation Figure 9.5b

15. DNA can be inserted into a cell by: • Microinjection • Gene gun Figure 9.6 & 7

16. Obtaining DNA • Gene libraries are made of pieces of an entire genome stored in plasmids or phages • cDNA is made from mRNA by reverse transcriptase • Synthetic DNA is made by a DNA synthesis machine

17. Genetic Engineering: Cloning a Gene Figure 9.11.1

18. Genetic Engineering Figure 9.11.2

19. Making a Gene Product: Colony Hybridization Figure 9.12.1

20. Making a Gene Product: Colony Hybridization Figure 9.12.2

21. Therapeutic Applications • Subunit vaccines • Nonpathogenic viruses carrying genes for pathogen's antigens as vaccines • Gene therapy to replace defective or missing genes • Human Genome Project • Nucleotides have been sequenced • Human Proteome Project may provide diagnostics and treatments

22. Scientific Applications • Understanding of DNA • Sequencing organisms' genomes • DNA fingerprinting for identification Figure 9.16

23. Southern Blotting Figure 9.15.1

24. Southern Blotting Figure 9.15.2

25. Southern Blotting Figure 9.15.3

26. Safety Issues and Ethics • Avoid accidental release • Genetically modified crops must be safe for consumption and for the environment • Who will have access to an individual's genetic information?