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Fig 11-1

Chapter 11: recombinant DNA and related techniques. Fig 11-1. Recombinant (chimeric) DNA: fused DNA from two different organisms Recombinant clone: vector (bacterial plasmid, virus) + insert (DNA fragment to be cloned) Recombinant (transgenic) organisms:

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Fig 11-1

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  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

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

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

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

  42. Fig 11-29

  43. Fig 11-30

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

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

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

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

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

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

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