1. Copyright © 2010 Pearson Education, Inc. Lectures prepared by Christine L. Case Chapter 9 Biotechnology and Recombinant DNA

2. Copyright © 2010 Pearson Education, Inc. -Compare and contrast biotechnology, genetic modification, and recombinant DNA technology. -Identify the roles of a clone and a vector in making recombinant DNA. -Compare selection and mutation. -Define restriction enzymes, and describe why they are used to make recombinant DNA. -List the four properties of vectors. -Describe the use of plasmid and viral vectors. -Provide an example of when PCR might be used -Describe five ways of getting DNA into a cell. -Describe how a genomic library is made. -Differentiate cDNA from synthetic DNA. -List one advantage of modifying each of the following: E. coli, Saccharomyces cerevisiae, mammalian cells, plant cells. -List and describe the different types and uses of biotechnology application, including Southern blotting, Diagram DNA fingerprinting for Microbial Forensics, Genetic engineering, and Nanotechonlogy Introduction to Biotechnology – Learning Objectives Upon completion of this chapter, you should be able to:

3. Copyright © 2010 Pearson Education, Inc. Biotechnology and Recombinant DNA  Biotechnology: The use of microorganisms, cells, or cell components to make a product.  Foods, antibiotics, vitamins, enzymes  Recombinant DNA (rDNA) technology: Insertion or modification of genes to produce desired proteins

4. Copyright © 2010 Pearson Education, Inc. Biotechnology and Recombinant DNA  Vector: Self-replicating DNA used to carry the desired gene to a new cell  Example: a plasmid  Clone: Population of cells arising from one cell, each carries the new gene

5. Copyright © 2010 Pearson Education, Inc. Figure 9.1 A Typical Genetic Modification Procedure

6. Copyright © 2010 Pearson Education, Inc. Figure 9.1 A Typical Genetic Modification Procedure

7. Copyright © 2010 Pearson Education, Inc. Table 9.2

8. Copyright © 2010 Pearson Education, Inc. Table 9.2

9. Copyright © 2010 Pearson Education, Inc. Table 9.3

10. Copyright © 2010 Pearson Education, Inc. -Selection of mutants -Restriction Enzymes -Recombinant DNA -Vectors -Plasmids -Viral vectors -Transposons -PCR Tools of Biotechnology

11. Copyright © 2010 Pearson Education, Inc. Selection and 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  Select and culture microbe with the desired mutation

12. Copyright © 2010 Pearson Education, Inc. Restriction Enzymes  Cut specific sequences of DNA  Destroy bacteriophage DNA in bacterial cells (bacteriophages are species specific)  Host DNA cannot be digested by restriction enzymes because of methylated cytosines ANIMATION: Recombinant DNA Technology

13. Copyright © 2010 Pearson Education, Inc. Table 9.1

14. Copyright © 2010 Pearson Education, Inc. Figure 9.2 Restriction Enzyme & Recombinant DNA

15. Copyright © 2010 Pearson Education, Inc. Vectors  Carry new DNA to desired cell  Shuttle vectors can exist in several different species (bacteria, mammalian cells, yeasts, etc.).  Plasmids and viruses can be used as vectors

16. Copyright © 2010 Pearson Education, Inc. Figure 9.3 A Plasmid Vector Used for Cloning

17. Copyright © 2010 Pearson Education, Inc. Polymerase Chain Reaction (PCR)  To make multiple copies of a piece of DNA enzymatically  Used to  Clone DNA for recombination  Amplify DNA to detectable levels  Sequence DNA  Diagnose genetic disease  Detect pathogens ANIMATION PCR: Components ANIMATION PCR: Overview

18. Copyright © 2010 Pearson Education, Inc. Figure 9.4 PCR

19. Copyright © 2010 Pearson Education, Inc. Figure 9.4 PCR

20. Copyright © 2010 Pearson Education, Inc. Figure 9.4 PCR ANIMATION PCR: Process

21. Copyright © 2010 Pearson Education, Inc. -Inserting DNA -Copying DNA -Making a product -Therapeutic Applications -Scientific Applications Techniques of Genetic Modification

22. Copyright © 2010 Pearson Education, Inc. Figure 9.5b Inserting Foreign DNA into Cells  DNA can be inserted into a cell by  Electroporation  Transformation  Protoplast fusion

23. Copyright © 2010 Pearson Education, Inc. Figure 9.5a Process of Protoplast Fusion

24. Copyright © 2010 Pearson Education, Inc. Inserting Foreign DNA into Cells  DNA can be inserted into a cell by  Gene gun  Microinjection

25. Copyright © 2010 Pearson Education, Inc. Figure 9.6 A Gene Gun

26. Copyright © 2010 Pearson Education, Inc. Figure 9.7 Microinjection of Foreign DNA

27. Copyright © 2010 Pearson Education, Inc. Figure 9.8 Obtaining DNA  Genomic libraries are made of pieces of an entire genome stored in plasmids or phages, which are then stored inside of a host

28. Copyright © 2010 Pearson Education, Inc. Figure 9.9 Obtaining DNA  Complementary DNA (cDNA) is made from mRNA by reverse transcriptase

29. Copyright © 2010 Pearson Education, Inc. Figure 9.10 Obtaining DNA  Synthetic DNA is made by a DNA synthesis machine

30. Copyright © 2010 Pearson Education, Inc. Figure 9.11 Selecting a Clone

31. Copyright © 2010 Pearson Education, Inc. Figure 9.11 Selecting a Clone

32. Copyright © 2010 Pearson Education, Inc. Figure 9.12 Selecting a Clone

33. Copyright © 2010 Pearson Education, Inc. Figure 9.12 Selecting a Clone

34. Copyright © 2010 Pearson Education, Inc. Making a Product 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 Figure 9.13

35. Copyright © 2010 Pearson Education, Inc. Making a Product Saccharomyces cerevisiae  Used because it is easily grown and its genomics are known  May express eukaryotic genes easily Mammalian cells  May express eukaryotic genes easily  Harder to grow Plant cells and whole plants  May express eukaryotic genes easily  Plants easily grown

36. Copyright © 2010 Pearson Education, Inc. Therapeutic Applications of rDNA  Producing human enzymes and other proteins  Creating Subunit vaccines (only contain a protein portion)  Nonpathogenic viruses carrying genes for pathogen's antigens as DNA vaccines  Gene therapy to replace defective or missing genes  Gene silencing  Identifying entire genomes  Using shotgun sequencing  The human genome project

37. Copyright © 2010 Pearson Education, Inc. Figure 9.14 RNA Interference (RNAi) – a type of gene silencing

38. Copyright © 2010 Pearson Education, Inc. Figure 9.15 Random Shotgun Sequencing

39. Copyright © 2010 Pearson Education, Inc. The Human Genome Project  Nucleotides have been sequenced  Human Proteome Project may provide diagnostics and treatments  Reverse genetics: Block a gene to determine its function

40. Copyright © 2010 Pearson Education, Inc. Figure 9.17 Scientific Applications  Understanding DNA  Sequencing organisms' genomes  DNA fingerprinting for identification

41. Copyright © 2010 Pearson Education, Inc. Figure 9.16 Southern Blotting

42. Copyright © 2010 Pearson Education, Inc. Figure 9.16 Southern Blotting

43. Copyright © 2010 Pearson Education, Inc. Figure 9.16 Southern Blotting

44. Copyright © 2010 Pearson Education, Inc. Forensic Microbiology  PCR  Primer for a specific organism will cause application if that organism is present  Real-time PCR: Newly made DNA tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle  Reverse-transcription (RT-PCR): Reverse transcriptase makes DNA from viral RNA or mRNA

45. Copyright © 2010 Pearson Education, Inc. Example: A Norovirus Outbreak  Are the outbreaks related?  What is the source? Clinical Focus, p. 266

46. Copyright © 2010 Pearson Education, Inc. Nanotechnology  Bacteria can make molecule-sized particles or they can provide the much needed metals to construct the tiny machinery necessary in nanotechnology Figure 9.18

47. Copyright © 2010 Pearson Education, Inc. Using Agrobacterium  Bt toxin  Herbicide resistance  Suppression of genes  Antisense DNA  Nutrition  Human proteins Figure 9.19

48. Copyright © 2010 Pearson Education, Inc. Using Agrobacterium Figure 9.20

49. Copyright © 2010 Pearson Education, Inc. Safety Issues and Ethics of Using rDNA  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?