Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) is a technique used for the amplification and identification of DNA or RNA. Also see mRNA (including transcriptase). It allows scientists to produce many millions of copies of a certain DNA sequence in a couple of hours.
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
Typically these steps are repeated in a cycle about 30 times generating a large amount of identical DNA copies. Therefore, PCR is often used just before doing an electrophoresis.
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 ends of the DNA region of interest. Included in the PCR reaction must be both the forward and reverse primers, in addition to Taq Polymerase and the DNA template. Alongside these substances the following must also be added; Magnesium Chloride, free nucleotides (dATP, dCTP, dTTP and dGTP) and a Tris-HCl (pH 8.0) buffer.
PCR is carried out in a thermal cycler and the enzyme 'Taq Polymerase' (isolated from Thermus 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.
When a PCR machine is unavailable a water bath can be used instead.
- ↑ Hartl D. L., Ruvolo M. (2012), Genetics: Analysis of genes and genomes, Eight Edition, Jones & Bartlett learning (Chapter 2 DNA Structure and Genetic Variation)
- ↑ http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.html