Polymerase Chain Reaction (PCR)

• PCR is a technique used to create many copies of a piece of DNA in vitro (outside the body of an organism). This process amplify a specific sequence of DNA millions of times in a few hours.
• Requirements for PCR:

  • Sequence of DNA (to act as a template)
  • Primers (Remember: these are required to start replication)
  • Nucleotides
  • Heat tolerant DNA polymerase (a special form of DNA polymerase that can act at high temperatures - optimum 70°C.)
• Primers in PCR

  • During PCR primers are used.

  • The primer used is selected to be

  complementary to a specific target sequence

  at the 3’ end of the DNA to be replicated.

  • This allows PCR to be highly specific to a

  particular sequence of DNA.
• PCR – Cycle 1

  • The process of PCR can be described as ‘thermal

  cycling’ as the DNA is repeatedly heated and cooled.

  • The DNA is heated to between 920C and 98 0C to break

  the hydrogen bonds between base pairs. This allows

  for the separation of the two strands of DNA.

  • The DNA is then cooled to between 500C and 65 0C to

  allow the primers to bind to the target sequences on

  the single strands of DNA at their 3’ ends.
• PCR - Cycle 1 (cont'd)

  • The DNA is then heated to between 70 degrees celcius and 80 degrees celcius to

  allow the heat-tolerant DNA polymerase to replicate

  the region of DNA.

  • The product is two identical copies of DNA at the end

  of the first cycle.
• PCR – Cycle 2 e.t.c

  • The cycle is repeated using the two strands

  produced in cycle one.

  • The product of cycle two is therefore four

  identical copies of DNA.

  • This process is then repeated many times

  (around 20-30 times) with each cycle doubling

  the quantity of DNA present at the start of

  that cycle.
• It is now possible to use PCR on a large scale thanks to

  the development of a computerised thermal cycler, a

  machine that can carry out and control the repetitive

  temperature changes required.
• PCR has revolutionised research in many areas of Science. It has been used to amplify DNA from many sources such as:

  • Blood, semen or tissue from a crime scene for DNA fingerprinting.

  • Embryonic cells for prenatal diagnosis of genetic disorders (e.g. Deuchenne Muscular Dystrophy), Paternity testing.

  • Viruses

  • Preserved remains of extinct species

  • Chloroplasts for investigating plant evolution
• The following steps occur during the Polymerase Chain Reaction (PCR):

  1. Heating of sample of DNA
  2. Separation of DNA strands
  3. Binding of Primer
  4. Replication of DNA
• 1. DNA is heated to between 92 degrees celsius and 98 degrees celsius to break the hydrogen bonds between bases and separate the 2 strands
• 2. DNA is cooled to between 50 degrees celsius and 65 degrees celsius to allow primers to bind to target sequences.
• 3. It is then heated to between 70 degrees celsius and 80 degrees celsius for heat tolerant DNA polymerase to replicate the region of DNA.
• Primers are short strands of nucleotides which are complementary to specific target sequences at the two ends of the region of DNA to be amplified.