IN VITRO CLONING

• METHOD OF IN VITRO CLONING?

  polymerase chain reaction (PCR)
• REQUIREMENTS FOR PCR?

  • DNA fragment
  • DNA polymerase
  • primers
  • nucleotides
  • thermocycler
• PURPOSE OF DNA POLYMERASE?

  • enzyme
  • catalyses formation of phosphodiester bonds
  • between nucleotides
  • forms sugar-phosphate backbone
• PURPOSE OF PRIMERS?

  • short nucleotide sequence
  • set of bases complementary to those at one of the ends of each of the two DNA fragments
• STAGES OF PCR?

  STAGE 1 - SEPARATION

  • 95 degrees Celsius to break hydrogen bonds
  • high temperature
  • breaks hydrogen bonds between complementary base pairs
  • DNA strands separate

  STAGE 2 - ANNEALING

  • 55 degrees Celsius to form new hydrogen bonds between primers and DNA
  • add primers
  • bind to end of both DNA strands by complementary base pairing

  STAGE 3 - ELONGATION

  • 72 degrees Celsius as DNA polymerase optimum
  • add nucleotides
  • add DNA polymerase
  • nucleotides bind to DNA strand by complementary base pairing
  • DNA polymerase causes phosphodiester backbone formation
  • new DNA strands
• EVALUATION OF IN VITRO CLONING?

  + rapid (1 cycle = 2 minutes/ 25 cycles = 1 million copies)

  + not require living cells

  -contamination
• EVALUATION OF IN VIVO CLONING?

  + useful in transformation

  + no contamination

  + accurate

  + cuts out specific genes

  + bacteria produce large quantities of gene products