1. Fig 11-1 Chapter 11: recombinant DNA and related techniques

2. Recombinant (chimeric) DNA: fused DNA from two different organisms Recombinant clone: vector (bacterial plasmid, virus) + insert (DNA fragment to be cloned) Recombinant (transgenic) organisms: host genome + clone from another organism

3. cDNA: “complementary DNA”; DNA complementary to RNA Usually made against mRNA cDNA is essentially an intron-less copy of a gene, minus 5’ and 3’ flanking regulatory regions of the gene Prepared using reverse transcriptase (an RNA- dependent DNA polymerase enzyme of RNA viruses)

4. Fig 11-2 Creating cDNA (DNA complementary to mRNA)

5. Fig 11-2 Creating cDNA (DNA complementary to mRNA)

6. Fig 11-2 Creating cDNA (DNA complementary to mRNA)

7. Fig 11-2 Creating cDNA (DNA complementary to mRNA) Creates clonable DNA copy of specific mRNA or can make cDNA library (representing mRNA population)

8. Fig 11-3 Using restriction sites to create a recombinant molecule

9. Fig 11-4

10. 4 -6 4 -4 pallindromic sequence cohesive ends

11. Fig 11-5 Using restriction sites to create a recombinant molecule

12. Fig 11-5 Using restriction sites to create a recombinant molecule

13. Fig 11-6 Cells receiving a complete plasmid form colony Grow and purify DNA from single colony Useful for inserts <10kb

14. Fig 11-6 Using antibiotic resistance markers to select plasmid-bearing colonies

15. Bacteriophage lambda: engineered as vector for cloning large DNA fragments Central 1/3 of genome (~45 kb) contains lysogenic function genes Can substitute ~15 kb cloned DNA into genome and the virus is still capable of lytic infection e.g., the Drosophila genome (~150,000 kb) can be contained in a minimum of 10,000 recombinant lambda clones (can fit on one 15 cm Petri plate)

16. Fig 11-7 Creating a genomic library in bacteriophage lambda

17. Fig 11-7 Creating a genomic library in bacteriophage lambda Useful for inserts 10-20kb

18. Fig 11-8 Useful for inserts 100-300kb

19. Fig 11-9

20. Identifying a desired clone/gene in a library: Use a probe (previously cloned DNA, oligonucleotide, or antibody)

21. Fig 11-11 Detecting & isolating a specific clone within a library by hybridization

22. Fig 11-1 Using an antibody to detect & isolate a specific clone within a library

23. Identifying a desired clone/gene in a library: Use a probe (previously cloned DNA, oligonucleotide, or antibody) Functional complementation (useful in organisms with small genomes) Positional cloning (chromosome “walk” to mutant rearrangement site)

24. Fig 11-15 Chromosome walking to identify/isolate a region containing a gene

25. Fig 11-13 Agarose gel electrophoresis separates DNA fragments by size: restrict cloned DNA electrophoresis stain with ethidium bromide visualize under UV

26. Fig 11-14 Southern/Northern blot analysis agarose gel electrophoresis transfer to nitrocellulose hybridize with radioactive probe autoradiograph to detect bands containing probe sequence

27. Fig 11-16 Using restriction sites as markers to map a DNA fragment

28. Fig 11-16 Using restriction sites as markers to map a DNA fragment

29. Fig 11-17 Dideoxynucleotide used for Sanger DNA sequencing

30. Fig 11-18 Sanger dideoxy DNA sequencing

31. Fig 11-18 Sanger dideoxy DNA sequencing Mixture of ddATP + dATP permits formation of chains of various lengths common 5’ end (primer) vary by 3’ ends, marking locations of A residues (T residues on template)

32. Fig 11-18 Sanger dideoxy DNA sequencing

33. Fig 11-18 Sanger dideoxy DNA sequencing

34. Fig 11-19 Automated sequencing readout of Sanger dideoxy DNA sequencing

35. Fig 11-20 An initial bioinformatic analysis Scan sequence for exceptionally long ORFs

36. Polymerase chain reaction (PCR) Uses heat-stable DNA polymerase (e.g., Taq polymerase) Requires two opposite-strand primers; ~100 bp - ~3 kb apart on the target template Uses a regimen of temperature cycling to amplify the DNA target between the two primers

37. Fig 11-21 Polymerase chain reaction Specific primers permit specific amplification of a DNA segment

38. Fig 11-22 Understanding alkaptonuria

40. Fig 11-24 Detecting sickle-cell β–globin allele

41. Fig 11-24 Detecting sickle-cell β–globin allele Heterozygote?

42. Fig 11-28 Ti plasmid: a vehicle for making transgenic plants

43. Fig 11-29

44. Fig 11-30

45. Fig 11-31 Inherited as a Mendelian dominant marker

46. Engineering of mammalian genomes Insert a gene (relatively easy) Destroy a gene (“knockout”) Replace a gene (e.g., gene therapy)

47. Insertions at random (ectopic) sites Ectopic transformation of mouse embryos Fig 11-34

48. Making a targeted mutation (“knockout”) in mouse cells Fig 11-35

49. Making a targeted mutation (“knockout”) in mouse cells Fig 11-35

50. Making a targeted mutation (“knockout”) in mouse cells Fig 11-35

51. Fig 11-36 Using embryonic stem cells to make a knockout mouse

52. Fig 11-36 Using embryonic stem cells to make a knockout mouse

53. Gene replacement therapy of lit mice Fig 11-38

54. Gene replacement therapy of lit mice Fig 11-38

55. Complications arising with germline gene therapy to cure genetic diseases in mammals is that most transgene integration events are random (not targeted) Transgene does not replace defective gene (just complements it) Transgene insert might disrupt another gene (creating an undesired mutation) Transgene will usually segregate independently from the disease-causing gene

56. Alternatives in gene therapy Fig 11-39 e.g., transgene on viral vector

57. Fig 11-