1. Today: Biotechnology Exam #2 W 7/9 in class

2. Comparative genomics also has been used to identify recently mobilized transposons in genetically diverse humans. For example, over 600 recent transposon insertions were identified by examining DNA resequencing traces from 36 genetically diverse humans. Tbl 1 Which transposable elements are active in the human genome? (2007) Ryan E. Mills et al. Trends in Genetics 23: 183-191

3. DNA fingerprinting using RFLPs

4. Visualizing differences in DNA sequence by using restriction enzymes Sequence 1 Sequence 2

5. Restriction Enzymes cut DNA at specific sequences Fig 18.1

6. Examples of some restriction enzymes… tbl 18.3

7. Visualizing differences in DNA sequence by using restriction enzymes Sequence 1 Sequence 2 Fig 20.5+.6

8. Separating DNA on a gel by size Fig 20.6

9. Gel electrophoresis Fig 24.21

10. Visualizing differences in DNA sequence by RFLPs Fig 22.23

11. DNA fingerprinting

14. Can DNA be obtained from hair?

15. How can DNA be obtained from such a small sample?

16. The inventor of PCR

17. Polymerase Chain Reaction: amplifying DNA Fig 18.6

18. Polymerase Chain Reaction Fig 18.6

19. Polymerase Chain Reaction: Primers allow specific regions to be amplified. Fig 18.6

20. The inventor of PCR PCR animation http://www.dnalc.org/ddnalc/resources/pcr.htmlhttp://www.dnalc.org/ddnalc/resources/pcr.html

21. Areas of DNA from very small samples can be amplified by PCR, and then cut with restriction enzymes for RFLP analysis.

22. Genetic Engineering: Direct manipulation of DNA Fig 18.2

23. Bacteria can be modified or serve as intermediates Fig 18.2

24. a typical bacteria Bacterial DNA plasmid DNA

25. A typical bacterial plasmid used for genetic engineering tbl 18.2

26. Moving a gene into bacteria via a plasmid Fig 18.2

27. Bacterial DNA plasmid DNA What problems exist for expressing eukaryotic gene in bacteria?

28. Reverse transcriptase can be used to obtain coding regions without introns. Fig 18.4

29. After RT, PCR will amplify the gene or DNA Fig 18.6

30. Moving a gene into bacteria via a plasmid RT and PCR Fig 18.2

31. Restriction Enzymes cut DNA at specific sequences Fig 18.1

32. Restriction enzymes cut DNA at a specific sequence Fig 18.1

33. Cutting the plasmid and insert with the same restriction enzyme makes matching sticky ends Fig 18.1

34. A typical bacterial plasmid used for genetic engineering

35. Using sticky ends to add DNA to a bacterial plasmid Fig 18.1

36. If the same restriction enzyme is used for both sides, the plasmid is likely to religate to itself. Fig 18.1

37. The plasmid is treated with phosphatase to remove the 5’-P, preventing self- ligation Fig 18.1

38. Transformation of bacteria can happen via several different methods. tbl 6.1

39. Bacteria can take up DNA from the environment Fig 9.2

40. Tbl 6.1 Transformation of bacteria can happen via several different methods all involving perturbing the bacterial membrane: Electroporation Heat shock Osmotic Stress

41. How can you know which bacteria have been transformed, and whether they have the insert? Fig 18.1

42. Resistance genes allow bacteria with the plasmid to be selected. Bacteria with the resistance gene will survive when grown in the presence of antibiotic

43. Fig 20.5 Is the insert present? Plasmids with the MCS in the lacZ gene can be used for blue/white screening… Fig 18.1

44. A typical bacterial plasmid used for genetic engineering

45. Intact lacZ makes a blue color when expressed and provided X-galactose

46. When the lacZ gene is disrupted, the bacteria appear white

47. Blue/white screening: Transformed bacteria plated on antibiotic and X- gal plates. Each colony represents millions of clones of one transformed cell. Fig 18.1

48. Successful transformation will grow a colony of genetically modified bacteria Fig 18.1

49. Inserting a gene into a bacterial plasmid RT and/or PCR Fig 18.1

50. Millions of Hectares Texas = 70 ha Bacteria can be used to transform plants Global area planted with GM crops http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html

51. Agrobacterium infect plants, inserting their plasmid DNA into the plants genome. Fig 19.15b

52. Fig 19.15 Agrobacterium infect plants, inserting their plasmid DNA into the plants genome.

53. By replacing the gall forming genes with other DNA when the Agrobacterium infect a plant, it will insert that DNA into the plant. Fig 19.16

54. The generation of a transgenic plant Grown on herbicide

55. How do you know whether the gene you want to express has the correct sequence? DNA sequencing *Not covered in class; will NOT be on the exam; check it out if you are interested.*

56. The structure of 2 ’,3 ’ -dideoxynucleotides Fig 18.14 *Not covered in class; will NOT be on the exam; check it out if you are interested.*

57. Fig 18.15 The dideoxy sequencing method *Not covered in class; will NOT be on the exam; check it out if you are interested.*

58. Fig 18.15 The dideoxy sequencing method *Not covered in class; will NOT be on the exam; check it out if you are interested.*

59. Gel produced by the dideoxy sequencing method *Not covered in class; will NOT be on the exam; check it out if you are interested.*

60. Fig 18.16 Computerized sequencers use a similar method *Not covered in class; will NOT be on the exam; check it out if you are interested.*

61. …now enjoy making Frakencritters.

62. What is the largest scale experiment that is being currently performed?

63. Genetically Modified Organisms

64. Human resource production and use Worldwide Grain Production per Person http://www.earth-policy.org/Updates/2008/Update69.htm

65. World Grain Stocks as Days of Consumption http://www.earth-policy.org/Updates/2008/Update69.htm

66. ~60 percent of the world grain harvest is consumed as food, ~36 percent as feed, and ~3 percent as fuel. While the use of grain for food and feed grows by roughly 1 percent per year, that used for fuel is growing by over 20 percent per year. http://www.earth-policy.org/Updates/2008/Update69.htm

67. Wheat prices Corn prices late 1999 - Dec 2007

68. http://www.earth-policy.org/Updates/2008/Update69.htm

69. Increasing oil prices drive up potential profit, and cost of food crops Cost of oil----Profitable cost of corn for ethanol $50 ---- $4/bushel $100 ---- $7/bushel $140 ---- $10/bushel Current corn price: ~$8/bushel http://www.farmdoc.uiuc.edu/manage/newsletters/fefo07_11/fefo07_11.html http://www.earth-policy.org/Updates/2008/Update69.htm

70. Whereas previous dramatic rises in world grain prices were weather-induced, this one is policy-induced and can be dealt with by policy adjustments. If the entire U.S. grain harvest were converted into ethanol, it would satisfy scarcely 18 percent of our automotive fuel needs. http://www.earth-policy.org/Updates/2008/Update69.htm

71. Land use in USA, 1997

72. Hunter- Gatherer Agricultural Industrial Artificial Selection (breeding)

73. Modern cows were bred from earlier bovine species.

74. from TeosinteMaize By artificial selection that began ~10,000 years ago.

76. Different foods from one species of plant. Created by artificial selection.

77. Wild member of Brassica oleracea: small side buds Brussels sprouts: extremely large side buds Artificial selection Number of Individuals Size of buds (cm) 0123 1. Select individuals that have the largest side buds and breed them. 2. Of the offspring, select individuals that have the largest side buds and breed them. 3. Of the offspring, select individuals that have the largest side buds and breed them. 4. After several generations, bud size increases dramatically.

78. Hunter- Gatherer Agricultural Industrial increased technology and understanding of molecular biology

79. Genetic Engineering: Direct manipulation of DNA Fig 18.1

80. Millions of Hectares Texas = 70 ha Global area planted with GM crops http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html

82. Why Change a Plant’s DNA? Can change plant so that it has new or different characteristics –Produce needed protein –Insect resistance (Bt toxin) –Herbicide resistance (Round-up) –Drought or other stress resistance Tbl 19.5

83. % of Total US Acres Common GM Crops in the U.S. http://blog.wired.com/wiredscien ce/2007/09/mon santo-is- hap.html

84. Herbicide Tolerance; Insect Resistance; Viral Resistance Types of Genetically Modified Crops

85. % of Total US Acres Common GM Crops in the U.S. http://blog.wired.com/wiredscien ce/2007/09/mon santo-is- hap.html

86. Millions of Hectares Texas = 70 ha Is changing an organsisms DNA a good idea? http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html

87. Is Changing an Organism’s DNA a Good or Bad Idea? Genes flow within species and between species. Are GM foods safe for the environment? Are GM foods profitable for: –Consumers, Farmers, and Companies?

88. AAL 25.17 Pollution in Gulf of Mexico from run-off in the Mississippi river

89. Pollen is easily transferred from plant to plant, and to related species.

90. GM crops illegal GM crops found in Oaxaca and other MX states Genetic information moves.

91. GM crops illegal GM crops found in Oaxaca and other MX states Genetic information moves, and is self-replicating.

92. Many Questions, but Few Answers? Are herbicide or insect resistant plants safe to eat? Are GM foods safe for the environment? Are GM foods profitable for: –Consumers, Farmers, and Companies?

93. Herbicide Tolerance; Insect Resistance; Viral Resistance Types of Genetically Modified Crops

94. Patents give 17 year monopoly for inventor

95. Examples of current patents: Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties

96. Examples of current patents: Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties Evolutec has patents on proteins in tick saliva for use as anti-inflammatory

97. Examples of current patents: Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties Evolutec has patents on proteins in tick saliva for use as anti-inflammator GTG in Australia has patents on non-coding human DNA for detecting risk of various diseases

98. Examples of current patents: Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties Evolutec has patents on proteins in tick saliva for use as anti-inflammatory GTG in Australia has patents on non-coding human DNA for detecting risk af various diseases Harvard patented cancer susceptible mouse

99. Examples of current patents: Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties Evolutec has patents on proteins in tick saliva for use as anti-inflammatory GTG in Australia has patents on non-coding human DNA for detecting risk af various diseases Harvard patented cancer susceptible mouse Over 70 U.S. patents for use of Indian neem tree

100. Neem, say the women, helps babies sleep, keeps flies away, is a cosmetic, a disinfectant and a pesticide. Its leaves make good cattle fodder, its twigs are good for teeth and gums. It is used, they say, for snake bites, malaria, hysteria, high blood pressure, pain relief, skin diseases and a host of other ailments. Over 70 U.S. patents for use of Indian neem tree

101. Neem, say the women, helps babies sleep, keeps flies away, is a cosmetic, a disinfectant and a pesticide. Its leaves make good cattle fodder, its twigs are good for teeth and gums. It is used, they say, for snake bites, malaria, hysteria, high blood pressure, pain relief, skin diseases and a host of other ailments. Over 70 U.S. patents for use of Indian neem tree One was rejected due to previous use in indigenous Indian culture

102. Concerns of developing nations vs. industrialized countries Bioprospecting

103. Lack of research on third world problems. (tuberculosis, malaria, cholera, dysentery… Not profitable.

104. Where does the information about genetically modified foods come from?

105. Millions of Hectares Texas = 70 ha Global area planted with GM crops http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html

106. Today: Biotechnology Exam #2 W 7/9 in class