1. Today: Biotechnology
Exam #2 Th 10/23 in class

2. Map of human chromosome 20
Your DNA
Map of human chromosome 20

3. Over 600 recent transposon insertions were identified by examining DNA 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:

4. DNA fingerprinting using RFLPs

5. Visualizing differences in DNA sequence by using restriction enzymes

6. Restriction Enzymes cut DNA at specific sequences
Fig 18.1
Restriction Enzymes cut DNA at specific sequences

7. Examples of some restriction enzymes…
tbl 18.3
Examples of some restriction enzymes…

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

9. Separating DNA on a gel by size
Fig 20.6
Separating DNA on a gel by size

10. Fig 24.21
Gel electrophoresis

11. The different sized bands can arise from different cut sites and/or different number of nucleotides between the cut sites.
Sequence 1
Sequence 2
Sequence 1
Fig 22.23
Sequence 2

12. DNA fingerprinting

13. DNA fingerprinting

14. DNA fingerprinting

15. Can DNA be obtained from hair?

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

17. The inventor of PCR

18. Polymerase Chain Reaction: amplifying DNA
Fig 18.6
Polymerase Chain Reaction:
amplifying DNA

19. Polymerase Chain Reaction
Fig 18.6
Polymerase Chain Reaction

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

21. The inventor of PCR
PCR animation

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

23. Genetic Engineering: Direct manipulation of DNA
Fig 18.2

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

25. a typical bacteria
Bacterial DNA
plasmid DNA

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

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

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

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

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

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

32. Restriction Enzymes cut DNA at specific sequences
Fig 18.1
Restriction Enzymes cut DNA at specific sequences

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

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

35. A typical bacterial plasmid used for genetic engineering

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

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

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

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

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

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

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

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

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

45. A typical bacterial plasmid used for genetic engineering

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

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

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

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

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

51. Bacteria can be used to transform plants
Global area planted with GM crops
Texas = 70 ha
Millions of Hectares

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

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

54. 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

55. The generation of a transgenic plant
Fig 19.16
The generation of a transgenic plant
Grown on herbicide