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  1. 1. PCR Polymerase Chain Reaction
  2. 2. Invented by Kary Mullis Mullis and Faloona, 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Nobel Prize 1993
  3. 3. Kary Mullis himself….
  4. 4. “I was working for Cetus, making oligonucleotides. They were heady times. Biotechnology was in flower and one spring night while the California buckeyes were also in flower I came across the polymerase chain reaction. I was driving with Jennifer Barnett to a cabin I had been building in northern California. She and I had worked and lived together for two years. She was an inspiration to me during that time as only a woman with brains, in the bloom of her womanhood, can be. That morning she had no idea what had just happened. I had an inkling. It was the first day of the rest of my life.” - from Karry Mullis’s autobiography at the Nobel e-Museum
  5. 5. PCR Specifically targets and amplifies a SINGLE sequence from within a complex mixture of DNA. How is this different from cloning?
  6. 6. Takes advantage of basic requirements of replication A DNA template Nucleotides Primers polymerase PCR is DNA replication in a test tube
  7. 7. Primers Must have some information about sequence flanking your target Primers provide specificity
  8. 8. 5’ 3’ 3’ 5’ Complementary to opposite strands with 3’ ends pointing towards each other Should have similar melting temperatures Be in vast excess
  9. 9. Melting temperature TmoC = 2(A/T) + 4(G/C) TmoC Temperature at which half possible H bonds are formed
  10. 10. The basic process dsDNA Denature (95 degrees)
  11. 11. 5’ 3’ 3’ 5’
  12. 12. Thermocycling 94 degrees 55 degrees 70 degree
  13. 13. Heat-stable polymerase is vital to the ease of the process…
  14. 14. Thermus aquaticus:
  15. 15. The Thermus aquaticus DNA polymerase Taq Not permanently destroyed at 94ºC Optimal temperature is 72ºC
  16. 16. Problems with Taq Does not have proof readng ability Error rate 1 in 2 X 104 bases Seems rare but can be recovered in cloning a single molecule Newer polymerases have high fidelity
  17. 17. Termplates for PCR Small amount of template In theory a single molecule Do not need to isolate sequence of interest DNA template need not be highly purified DNA is stable in absence of nucleases
  18. 18. Templates for PCR Dried blood Semen stains
  19. 19. Templates for PCR Dried blood Semen stains Vaginal swabs Single hair Fingernail scrapings Insects in Amber Egyptian mummies Buccal Swab Toothbrushes
  20. 20. PCR variations Add 5’ extensions for cloning 3’ 5’ 3’ 5’ 5’ G A A T T 3’ C 3’ C T T A A G 5’
  21. 21. Cloning PCR Fragments Taq leaves 3’ A overhang. A A T T
  22. 22. rtPCR Reverse trancriptase PCR Use mRNA as a template Isolated cDNA clones Can be quantitative
  23. 23. Inverse PCR unknown known unknown Known unknown
  24. 24. known unknown
  25. 25. Nested primers PCR primers are not always an exact match! Degeneracy Lower annealing temperatures increase chances of amplifying something! Could be wrong thing!
  26. 26. Nested primers 2 1 2 1
  27. 27. Quantitative PCR
  28. 28. Real Time PCR Detection and quantitation of fluorescent reporter the signal of which increases in direct proportion to the amount of PCR product in a reaction Does not measure the amount of end product but its production in real time
  29. 29. SYBR green Also binds primer dimers Can overestimate product
  30. 30. Molecular Beacons Uses FRET Fuorescence Resonance Energy Transfer Uses two sequence specific oligonucleotides labeled with fluorescent dyes
  31. 31. Taq Man
  32. 32. Other application of PCR Detection of mutations screen for inherited disorders Detection of HIV Not standard test given Detect tuberculosis without culturing Prenatal sex determination DZY1 = Y specific sequence present in 5000 copies
  33. 33. Other application of PCR Preimplantation diagnosis of genetic diseases Forensics Paternity testing
  34. 34. Forensics STR Short Tandem Repeats 2 to 7 base pairs repeated 7-40 times Replaced VNTRs in forensic analysis 13 Highly polymorphic loci have been selected by FBI Population match probabilities 0.1 - 0.28 Probability One in 5.7 X 10-15 Combined DNA Index System (CODIS)
  35. 35. STR analysis of family of last Tsar of Russia
  36. 36. VNTR’s Can use PCR to visualize VNTRs Eg. pMCT118 in chromosome 1
  37. 37. VNTR analysis
  38. 38. Problems with PCR Contamination Theoretically one molecule can amplify Takes one mismatch early on to amplify the wrong fragment