1. Genetic Engineering 1 Lecture 18 Pages 323 - 340

2. The Tools of Molecular Biology

3. 10_01_experiment.DNA.jpg Old fashioned way was to breed for what you wanted Mendel did it!

4. 10_02_cell_sorter.jpg

5. Once you have the cells what next? Issue was to examine the DNA in a consistent manner Best method is to use restriction enzymes –Come mainly from bacteria –Use individually or in a mix

6. 10_04_Restrict.nuclease.jpg

7. What do you do with these digested fragments of DNA? Isolate those that you want to work on How? –Best method is to use gel electrophoresis Agarose Polyacrylamide

8. 10_05_gel.electrophor.jpg

9. Then what do you do with this piece of DNA? Clone Sequence –Rely on the use of dideoxy nucleotides

10. 10_07_1_enzym.dideoxy.jpg

11. 10_07_2_enzym.dideoxy.jpg

12. 10_08_DNA.sequencing.jpg

13. 10_09_Shotgun.sequenc.jpg

14. 10_10_Repetit.sequence.jpg

15. 10_11_BAC.clones.jpg

16. 10_12_de_renaturation.jpg

17. 10_13_hybridization.jpg

18. Blotting Purpose to make a permanent record of the results of a gel electrophoresis. The compass - Southern blots - DNA Northern blots - RNA Western blots - Proteins

19. 10_14_1_Southrn.blotting.jpg

20. 10_14_2_Southrn.blotting.jpg

21. 10_15_DNA.microarrays.jpg

22. Cloning and growing One can use the techniques of cell biology to manufacture artificial and real products, be they genes, proteins, or organisms If you want to insert some DNA into another molecule then the best place to start is to use the same restriction enzyme to cut both - so they have the same ends.

23. 10_18_ DNA.in.vitro.jpg

24. 10_19_DNA.uptake.jpg Bacteria have the ability to ‘ingest’ DNA from their environment naturally. This property makes them able to change their properties very quickly - and dangerous to us.

25. 10_20_Bacteria.plasmid.jpg Bacteria are able to also pass between themselves, other small pieces of DNA known as plasmids. We can make use of plasmids to carry our test DNA into bacteria as shown on the next side…

26. 10_21_DNA ligase.jpg

27. 10_22_cloned.DNA.frag.jpg Small numbers of transformed bacteria can be grown to large numbers in simple growth media.

28. 10_23_genomic.library.jpg Genomic libraries of fragments of all human genes can be made by this technique. One can buy these libraries and use them to isolate any gene and grow that for experimental purposes. One can find the right cell using the technique on the next slide…

29. 10_24_hybridization.jpg

30. 10_25_cDNA.jpg Another technique is to use the mRNA from a cell to make DNA in the reverse direction. These DNA molecules represent just the genes that were active at the time the mRNA was recovered from the cell.

31. 10_26_Genomic_cDNA.jpg

32. 10_27_1_PCR_amplify.jpg

33. 10_27_2_PCR_amplify.jpg PCR - Polymerase Chain Reaction

34. 10_28_PCR_clones.jpg

35. 10_29_PCR_viral.jpg

36. 10_30_1_PCR_forensic.jpg

37. 10_30_2_PCR_forensic.jpg

38. 10_31_SerialDNA.clone.jpg

39. 10_32_expressionvector.jpg

40. 10_33_gene_protein.jpg

41. 10_34_Reporter.genes.jpg

42. 10_35_GFP.jpg

43. 10_36_mutagenesis.jpg

44. 10_37_engineered.org.jpg

45. 10_38_ES.cells.jpg

46. 10_39_Transgenic.mice.jpg

47. 10_40_Transgenic.plant.jpg