Fig 11-1

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

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

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

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

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

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

9. Fig 11-4

10. 4-6 4-4 pallindromic sequence cohesive ends Fig 11-4

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

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

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

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

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

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

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

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

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

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

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

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

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

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

30. Sanger dideoxy DNA sequencing Fig 11-18

31. 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) Fig 11-18

32. Sanger dideoxy DNA sequencing Fig 11-18

33. Sanger dideoxy DNA sequencing Fig 11-18

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

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

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

38. Understanding alkaptonuria Fig 11-22

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

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

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

43. Fig 11-29

44. Fig 11-30

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

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

47. Ectopic transformation of mouse embryos Insertions at random (ectopic) sites 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