IN VITRO CLONING

• METHOD OF IN VITRO CLONING?

  polymerase chain reaction (PCR)
• REQUIREMENTS FOR PCR?

  • DNA fragment
  • DNA (Taq) polymerase
  • primers
  • nucleotides
  • thermocycler
• PURPOSE OF DNA (Taq) POLYMERASE?

  • enzyme from thermophilic bacteria
  • 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
  • make DNA double stranded allowing DNA (Taq) polymerase to bind
  • prevents template strands from rejoining
• 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
• PROCESS OF PCR?

  1. Reaction mixture containing...

  • DNA fragments
  • primers
  • free DNA nucleotides
  • DNA polymerase

  1. Heat to 95

  • denatures DNA
  • breaks hydrogen bonds between bases
  • creates 2 template strands

  1. Cool to 55

  • primers anneal to start of template strand
  • complementary base pairing
  • hydrogen bonds

  1. Heat to 72

  • DNA nucleotides pair up with complementary bases
  • hydrogen bonds form (A/T, C/G)
  • new strand synthesised faster

  1. DNA polymerase joins nucleotides and makes sugar-phosphate backbone (condensation reaction, phosphodiester bonds)
  2. Repeat for other template = 2 DNA molecules
• IMPORTANCE OF KNOWING THE BASE SEQUENCE IN PCR?

  • for primers
  • to produce a complementary base sequence
  • to allow DNA (Taq) polymerase to bind
  • to prevent strands rejoining