Amplifying DNA fragments

In vivo… (incomplete)

In vitro

  • DNA fragments are amplified

  • copies of the DNA fragments are made outside of a living organism

  • Using the polymerase chain reaction (PCR)

  • PCR has several stages and is repeated over and over to make lots of copies

Denaturing

  1. A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase

  • primers are short pieces of DNA that are complementary to the bases at the start of the fragment you want

  • DNA polymerase is an enzyme that creates new DNA strands

  1. The DNA mixture is heated to 95°c to break the hydrogen bonds between the two strands of DNA

  2. The mixture is then cooled to between 50°c and 65°c so that the primers can bind/ anneal to the strands (optimum temp for enzymes)

Annealing

  1. The reaction mixture is heated to 72°c so DNA polymerase can work

  2. The DNA polymerase lines up free DNA nucleotides alongside each template and joins the nucleotides together. Specific base pairing means new complementary strands are formed

Elongating`

  1. Two new copies of the fragment of DNA are formed and one cycle of PCR is complete

  2. The cycle starts again with the mixture being heated to 95°c and this time all four strands (2 original, 2 new) are used as templates

  3. Each PCR cycle doubles the amount of DNA