Polymerase Chain Reaction (PCR)
PCR has three main stages:
1. Heat DNA to 95°C to melt the strands
2. Cool to 50 - 65°C to allow primers to anneal
3. Heat to 72°C to allow elongation
The most important part of the PCR reaction is the initial design of the primers. The primers are normally between 18 to 20 base pairs in length and must be completely complimentary to the DNA at the start of the region you are interested in amplyfing. Included in the experiment must be both the forward and reverse primers.
PCR is carried out in a thermal cycler and the enzyme 'Taq Polymerase' (isolated fromThermus aquaticus) is used as it is thermostable (is not denatured by high temperatures), therefore is not denatured at the high temperatures. Pfu (Pyrococcus furiosus) DNA Polymerase can also be used as it has better thermostability than Taq Polymerase and it possesses 3' to 5' proof reading activity.
The technique was developed by Kary Mulis in 1983 for which he was awarded the Nobel Prize in Chemistry in 1993.
PCR can be done using water baths at varying temperatures.