Today: Biotechnology

1. Today: Biotechnology

2. Over 600 recent transposon insertions were identified by examining DNA from 36 genetically diverse humans. Tbl 1Which 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. Fig 18.1 Restriction Enzymes cut DNA at specific sequences

6. tbl 18.3 Examples of some restriction enzymes…

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

8. Fig 20.6 Separating DNA on a gel by size

9. Fig 24.21 • Gel electrophoresis

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

11. DNA fingerprinting

12. DNA fingerprinting

13. 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. Fig 18.6 Polymerase Chain Reaction: amplifying DNA

18. Fig 18.6 Polymerase Chain Reaction

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

20. The inventor of PCR PCR animation http://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. tbl 18.2 A typical bacterial plasmid used for genetic engineering

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

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

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

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

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

31. Fig 18.1 Restriction Enzymes cut DNA at specific sequences

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

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

34. A typical bacterial plasmid used for genetic engineering

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

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

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

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

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

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

41. 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

42. A typical bacterial plasmid used for genetic engineering

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

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

45. 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.

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

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

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