Chapter 6: Sources of DNA: Polymerase Chain Reaction (PCR)

PCR allows for isolation of nucleic acids and analysis (can study rare genes by amplifying small amounts of DNA and generate specific fragments) also useful for cloning and RE site can be added to ends via primers

  • Requires: Template, Forward & Reverse Primers, dNTPs, DNA ligase, DNA polymerase, Buffer (Mg)
  • Initial denature: 2m, 95 (ensure ss), Denaturation: 30s, 95, Anneal: 30s, Tm - 5 (Tm=2*(AT)+4*(GC) Extend: 1m per 1000 bp, 72, Elongate: 5m, 72 Leave at 4oC
  • Annealing Conditions: Optimized for maximum binding of primer to template (computer program predict)
  • too low loses specificity, too high none would bind trial and error 40-60
  • Primer Design: Complementary to specific target sequence and not to each other, length of 12-30nt, melting temperature should be similar, avoid flappy (runs of AT/GC), modify (add nt, Re, fluorescent tag)
  • Thermostable Polymerase: Allows many cycles and keeps it active, higher proof reading, more processive and mutated from heat stable organisms, add blunt end using single A at end to clone into T hang vector
  • 4 dNTP: Usually unlabelled, for probe one is 32-P labelled or DIG
  • WCGW: no amplification (no primer / condition wrong) , amplification due to contaminant DNA
  • +ve Control: Known template to test conditions of the experiment
  • -ve Control: no template to test contamination
  • Benefits: Fast, sensitive, adaptable, cheap, use RNA/cDNA
  • Limitations: Sensitive = prone to contamination, must know sequence to design primers, difficult for long
  • Analyzing Results
  • Gel electrophoresis to look for expected size of PCR product
  • RE digest with RE known to cut the expected product
  • Sequence the product to verify it is correct (gold standard) important as it can identify mutations
  • Variations
  • RTPCR: cDNA template diluted so amount of product depends on starting cDNA template, intensity of product reflects abundance of mRNA, very sensitive to compare transcripts
  • Applications: Probe synthesis, cloning strategies, analysis of ligation produce, detection or splice variants

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