PCR

• SIMPLE DNA PROFILING

  1. isolate sample of DNA
  2. produce copies of DNA fragments in a sample using PCR
  3. carry out gel electrophoresis on DNA
  4. analyse resulting patterns of DNA fragments
• uses of PCR

  • produce large quantities of specific fragments of DNA or RNA sample
  • produce billions of identical copies of DNA or RNA
  • each cycle, DNA doubled
  • done in a thermal cycler (optimum temp)
• PCR needs

  • primers=short sequences of single stranded DNA that have complementary base sequences to the end of DNA or RNA being copied, defining the region being amplified
  • DNA polymerase=enzyme used to build new DNA or RNA strand, TAQ polymerase doesnt denature at high temps
  • free nucleotides= enables construction of new RNA or DNA strands
  • buffer solution= ensures optimal PH for reactions to occur
• PCR stages:

  1. denaturation- double stranded DNA heated to 95 degrees, breaks hydrogen bods between strands of DNA
  2. annealing- temp decreases to 50-60 degrees, primers can anneal to ends of single stranded DNA
  3. elongation- temp increases to 72 degrees, optimum temperature for DNA/TAQ polymerase to work at= complementary base pairing with free nucleotides, copy of sample created
• prep for gel electrophoresis

  • restriction enzyme= breaks DNA up into fragments of different lengths
  • fluorescent tags=enables DNA fragments to be seen under UV light