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PCR PPT

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  • 1. PCR Polymerase Chain Reaction
  • 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. Kary Mullis himself….
  • 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. PCR Specifically targets and amplifies a SINGLE sequence from within a complex mixture of DNA. How is this different from cloning?
  • 6. Takes advantage of basic requirements of replication A DNA template Nucleotides Primers polymerase PCR is DNA replication in a test tube
  • 7. Primers Must have some information about sequence flanking your target Primers provide specificity
  • 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. Melting temperature TmoC = 2(A/T) + 4(G/C) TmoC Temperature at which half possible H bonds are formed
  • 10. The basic process dsDNA Denature (95 degrees)
  • 11. 5’ 3’ 3’ 5’ http://www.dnalc.org/shockwave/pcranwhole.html
  • 12. Thermocycling 94 degrees 55 degrees 70 degree
  • 13. Heat-stable polymerase is vital to the ease of the process…
  • 14. Thermus aquaticus:
  • 15. The Thermus aquaticus DNA polymerase Taq Not permanently destroyed at 94ºC Optimal temperature is 72ºC
  • 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. 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. Templates for PCR Dried blood Semen stains
  • 19. Templates for PCR Dried blood Semen stains Vaginal swabs Single hair Fingernail scrapings Insects in Amber Egyptian mummies Buccal Swab Toothbrushes
  • 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. Cloning PCR Fragments Taq leaves 3’ A overhang. A A T T
  • 22. rtPCR Reverse trancriptase PCR Use mRNA as a template Isolated cDNA clones Can be quantitative
  • 23. Inverse PCR unknown known unknown Known unknown
  • 24. known unknown
  • 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. Nested primers 2 1 2 1
  • 27. Quantitative PCR
  • 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. SYBR green Also binds primer dimers Can overestimate product
  • 30. Molecular Beacons Uses FRET Fuorescence Resonance Energy Transfer Uses two sequence specific oligonucleotides labeled with fluorescent dyes
  • 31. Taq Man
  • 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. Other application of PCR Preimplantation diagnosis of genetic diseases Forensics Paternity testing
  • 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. STR analysis of family of last Tsar of Russia
  • 36. VNTR’s Can use PCR to visualize VNTRs Eg. pMCT118 in chromosome 1
  • 37. VNTR analysis
  • 38. Problems with PCR Contamination Theoretically one molecule can amplify Takes one mismatch early on to amplify the wrong fragment

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