PCR

• PCR - polymerase chain reaction
• three steps that repeat in cycles

  1. denaturation
  2. annealing
  3. extension
• PCR checklist

  • DNA template
  • Taq polymerase
  • free nucleotides
  • start and end primers
  • pH buffer
• 1. denaturation

  • heated to 95 °C
  • hydrogen bonds between DNA strands break
  • DNA now single stranded
• 2. annealing

  • cooled to 55 °C
  • hydrogen bonds can reform
  • DNA primers bind to complementary sequence on template
• 3. extension

  • heated to 72 °C
  • polymerase binds to primers, moves along
  • hydrogen bonds reform and phosphodiester bonds form
• primers

  • provides a double stranded section for DNA polymerase to bind and to add to
  • they set the boundaries for the region that will be amplified (only the 3' end is added to)
• Taq polymerase

  • from bacterium - thermus aquaticus
  • does not denature during step 1
  • can withstand high temperatures
  • can be used over many cycles
• Finding the total number of copies produced:

  T = D x 2C

  T = total number of copies produced

  D = number of DNA molecules at the beginning

  C = number of cycles of PCR done