Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) is a technique used for the amplification and identification of DNA or RNA of known sequence to give exponential products or copies. Also see mRNA (including transcriptase). It allows scientists to produce many millions of copies of a certain DNA sequence in a couple of hours. This technique was developed by an american biochemist Kary Mullis in 1984 .
PCR has three main stages:
- Strand Seperation : Heat dsDNA to 95°C for 15sto melt and seperate the strands.
- Hybridisation of Primer : Cool to 50 - 65°C to allow primers to anneal to the DNA strands.
- DNA Synthesis : 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. 18 to 20 base pairs for a primer are ideal because a 18-2 base sequence is quite unique and is therefore unlikely to be present in any other section other than the target DNA. This ensures that the primers bind only to the flanking sequences associated with the target DNA sequence. For shorter genomes a smaller primer can be used. Along with this included in the PCR reaction must be both the forward and reverse primers, in addition to Taq Polymerase (which requires MgCl2 for its effective activity) 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, which forms the core of PCR.Originally, DNA polymerase was added to the PCR reaction but it was denatured by the high temperatures, so had to be added at the end of every cycle. However, because Taq polymerase is thermostable, it isn't denatured so only needs to be added at the beginning of the reaction. 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.
PCR technique has many importance amongst which is it's use in identification of the orientation of cloned inserts, also, it is used in other fields such as forensic science- at crime scenes, science in general- to diagnosize diseases.
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 and Bartlett learning (Chapter 2 DNA Structure and Genetic Variation)
- ↑ Berg, J.M., Tymoczko, J.L. and Stryer, L. (2012). Biochemistry, 7 th Edition, New York , W.H.Freeman and Co Ltd. pg 151
- ↑ http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.html