Polymerase Chain Reaction (PCR)

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Polymerase Chain Reaction[1] (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 can amplify DNA without a host organism. 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 [2] for which he was awarded the Nobel Prize in Chemistry in 1993 .

PCR has three main stages:

  1. Strand Separation : Heat dsDNA to 95°C  for 15s to melt and separate the strands, denaturing the hydrogen bonds which hold them together.
  2. Hybridisation of Primer : Cool to 50 - 65°C to allow primers to anneal to the DNA strands. The temperature required at this stage is primer dependant.
  3. DNA Synthesis : Heat to 72°C to allow elongation. This extention is usually performed by free nucleotides with taq polymerase. 

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. At the end of he elongation phase, PCR products are stored at 4-10°C.

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. The primers are always found at the 5' ends of the final DNA fragments[3]. 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 for the logarithmic amplification to happen otherwise the amplification is only linear, 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, (a machine that is capable of varying temperature) when this is unavailable water baths can be used instead, and the enzyme 'Taq Polymerase' (isolated from Thermus aquaticus first isolated in 1976) 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. Pfu is from an organism of the archaea that lives in submarine vents, the enzmye survives 100°C.

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.

PCR is a very useful tool for scientists for several reasons. It can provide valuable diagnostic information in medicine as bacteria and viruses can easily be detected by the use of specific primers. For example, PCR can reveal the presence of a small amount of DNA from the human immunodeficiency virus (HIV) in persons who have not yet mounted an immune response to the pathogen. In these patients, assays designed to detect antibodies against the virus would yield a false negative test result. This is useful in the early diagnosis of HIV as well as helping to reduce the likelihood of further transmission.

There are an array of characteristics that makes PCR valuable in a clinical setting. Firstly, PCR is relatively inexpensive. Also, multiple copies can be made from samples which are old and degraded due to DNA being stable. The PCR reaction is also very quick, it only takes a few hours. PCR shows high sensitivity and specificity to even small pieces of DNA, the results of PCR can be made available in a short amount of time (a few hours usually) and it is a relatively cheap process at £1-2 per reaction [4].

Polymerase Chain Reaction first step is known as 'Strand Seperation. This is where you take the sequence of DNA you want to copy and heat it to 95oC. This causes the two strands of DNA to seperate as the Hyrdrogen Bonds between the bases break. The strands are heated for roughly 15 seconds. The next stage is reffered to as 'Hybridization of Primers' this is where primers are added to the DNA as it is cooled to 54oC to allow the hydrogen bonds to form between the primers and the DNA strands. The primers both bind the the 3' end of the template and complementary strands. The Primers are typically 20-30 nucleotides long. The third stages is called 'DNA Synthesis'. The solution containg the DNA and primers is heated to 72oC and the enzyme Taq polymerase is added. This specific enzyme is found at bacteria that live in hot springs, therefore it is able to function at this high temperature. Such organisms are said to be thermophiles. The Taq polymerase lengthens the DNA in the 5'-to-3' direction and takes place on both strands [5].


https://www.youtube.com/watch?v=Kx5qMjh-izA

References

  1. 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)
  2. Berg, J.M., Tymoczko, J.L. and Stryer, L. (2012). Biochemistry, 7 th Edition, New York , W.H.Freeman and Co Ltd. pg 151
  3. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2008) Molecular biology of the cell, 5th edition, New York: Garland Science. pg 544
  4. Berg, J.M., Tymoczko, J.L. and Stryer, L. (2012). Biochemistry, 7 th Edition, New York , W.H.Freeman and Co Ltd. pg 152
  5. Berg. J. M., Stryer. L. and Tymoczko. J.L. (2007) Biochemistry, 6th edition, New York : W. H. Freeman : Palgrave

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