1. Biotechnology and Recombinant DNA
Chapter 9
Biotechnology and Recombinant DNA

2. Q&A
Interferons are species specific, so that interferons to be used in humans must be produced in human cells. Can you think of a way to increase the supply of interferons so that they can be used to treat diseases?

3. Introduction to Biotechnology
Learning Objectives
9-1 Compare and contrast biotechnology, genetic modification, and recombinant DNA technology.
9-2 Identify the roles of a clone and a vector in making recombinant DNA.

4. 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

5. Biotechnology and Recombinant DNA
Vector: Self-replicating DNA used to carry the desired gene to a new cell
Clone: Population of cells arising from one cell, each carries the new gene

6. A Typical Genetic Modification Procedure
Figure 9.1

7. A Typical Genetic Modification Procedure
Figure 9.1

8. Table 9.2

9. Table 9.2

10. Table 9.3

11. Differentiate biotechnology and recombinant DNA technology. 9-1
In one sentence, describe how a vector and clone are used. 9-2

12. Tools of Biotechnology
Learning Objectives
9-3 Compare selection and mutation.
9-4 Define restriction enzymes, and outline how they are used to make recombinant DNA.
9-5 List the four properties of vectors.
9-6 Describe the use of plasmid and viral vectors.
9-7 Outline the steps in PCR, and provide an example of its use.

13. 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

14. Restriction Enzymes Cut specific sequences of DNA
Destroy bacteriophage DNA in bacterial cells
Cannot digest (host) DNA with methylated cytosines
ANIMATION: Recombinant DNA Technology

15. Table 9.1

16. Restriction Enzyme & Recombinant DNA
Figure 9.2

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

18. A Plasmid Vector Used for Cloning
Figure 9.3

19. 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: Overview
ANIMATION PCR: Components

20. PCR
Figure 9.4

21. PCR
Figure 9.4

22. PCR
ANIMATION PCR: Process
Figure 9.4

23. How are selection and mutation used in biotechnology? 9-3
What is the value of restriction enzymes in recombinant DNA technology? 9-4
What criteria must a vector meet? 9-5
Why is a vector used in recombinant DNA technology? 9-6
For what is each of the following used in PCR: primer, DNA polymerase, 94°C? 9-7

24. Techniques of Genetic Modification
Learning Objectives
9-8 Describe five ways of getting DNA into a cell.
9-9 Describe how a genomic library is made.
9-10 Differentiate cDNA from synthetic DNA.
9-11 Explain how each of the following is used to locate a clone: antibiotic-resistance genes, DNA probes, gene products.
9-12 List one advantage of modifying each of the following: E. coli, Saccharomyces cerevisiae, mammalian cells, plant cells.

25. Inserting Foreign DNA into Cells
DNA can be inserted into a cell by
Electroporation
Transformation
Protoplast fusion
Figure 9.5b

26. Process of Protoplast Fusion
Figure 9.5a

27. Inserting Foreign DNA into Cells
DNA can be inserted into a cell by
Gene gun
Microinjection

28. A Gene Gun
Figure 9.6

29. Microinjection of Foreign DNA
Figure 9.7

30. Obtaining DNA
Genomic libraries are made of pieces of an entire genome stored in plasmids or phages
Figure 9.8

31. Obtaining DNA
Complementary DNA (cDNA) is made from mRNA by reverse transcriptase
Figure 9.9

32. Obtaining DNA Synthetic DNA is made by a DNA synthesis machine
Figure 9.10

33. Selecting a Clone
Figure 9.11

34. Selecting a Clone
Figure 9.11

35. Selecting a Clone
Figure 9.12

36. Selecting a Clone
Figure 9.12

37. 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

38. Making a Product Saccharomyces cerevisiae Mammalian cells
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

39. Q&A
Interferons are species specific, so that interferons to be used in humans must be produced in human cells. Can you think of a way to increase the supply of interferons so that they can be used to treat diseases?

40. Contrast the five ways of putting DNA into a cell. 9-8
What is the purpose of a genomic library? 9-9
Why isn’t cDNA synthetic? 9-10
How are recombinant clones identified? 9-11
What types of cells are used for cloning rDNA? 9-12

41. Applications of rDNA Learning Objectives
9-13 List at least five applications of rDNA technology.
9-14 Define RNAi.
9-15 Discuss the value of the Human Genome Project.
9-16 Define the following terms: random shotgun sequencing, bioinformatics, proteomics.

42. Therapeutic Applications
Human enzymes and other proteins
Subunit vaccines
Nonpathogenic viruses carrying genes for pathogen's antigens as DNA vaccines
Gene therapy to replace defective or missing genes

43. RNA Interference (RNAi)
Figure 9.14

44. Random Shotgun Sequencing
Figure 9.15

45. 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

46. Explain how rDNA technology can be used to treat disease and to prevent disease. 9-13
What is gene silencing? 9-14
How are shotgun sequencing, bioinformatics, and proteomics related to the Human Genome Project? 9-15, 9-16

47. Applications of rDNA Learning Objectives
9-17 Diagram the Southern blotting procedure, and provide an example of its use.
9-18 Diagram DNA fingerprinting, and provide an example of its use.
9-19 Outline genetic engineering with Agrobacterium.

48. Scientific Applications
Understanding DNA
Sequencing organisms' genomes
DNA fingerprinting for identification
Figure 9.17

49. Southern Blotting
Figure 9.16

50. Southern Blotting
Figure 9.16

51. Southern Blotting
Figure 9.16

52. 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

53. Norovirus Outbreak Are the outbreaks related? What is the source?
Clinical Focus, p. 266

54. Norovirus Outbreak RT-PCR with a norovirus primer
Clinical Focus, p. 266

55. Nanotechnology
Bacteria can make molecule-sized particles
Figure 9.18

56. Using Agrobacterium Bt toxin Herbicide resistance Suppression of genes
Antisense DNA
Nutrition
Human proteins
Figure 9.19

57. Using Agrobacterium
Figure 9.20

58. What is Southern blotting? 9-17
Why do RFLPs result in a DNA fingerprint? 9-18
Of what value is the plant pathogen Agrobacterium? 9-19

59. Safety Issues and Ethics of Using rDNA
Learning Objective
9-20 List the advantages of, and problems associated with, the use of genetic modification techniques.

60. 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?

61. Identify two advantages and two problems associated with genetically modified organisms. 9-20