in vitro cloning

• Polymerase chain reaction is the artificial replication of short sequences of DNA , it uses artificial DNA primers to amplify a specific strand of DNA. Fragments of DNA undergo a series of heating and cooling steps in a thermocycler to replicate them many times.
• ingredients for PCR:

  1. DNA sample
  2. Nucleotides
  3. Enzyme; DNA polymerase sticks bases together to form a strand.
  4. Primers: a string of nucleotides bases complementary to the start and end of the DNA sequence to be amplified.
• In PCR the DNA polymerase used has a much higher optimum temperature than human polymerase.
• How it works:

  1. the mixture is heated to 94-96 degrees to break hydrogen bonds between the complimentary nucleotides base pairs and separate double stranded DNA into two single strands.
  2. The mixture is cooled to around 55-60 so that the primers can anneal to one end of each single strand of DNA which identifies where the polymerase should bind.
  3. The temperature is raised to 72 (optimum)for the polymerase. this enzyme catalyzes the addition of DNA nucleotides by complimentary base pairing to create two complementary double strands of DNA.
• The whole process is repeated again for many cycles. Use 1x2^n to find how many DNA stands.