Basic Polymerase Chain Reaction Techniques

New PCR technology is essential for improving human health and hence the quality of life of everyone on Earth. The new technology, which is being developed all the time, lets genetic scientists take specimens of genetic material and then copy the genetic sequence again and again. This can generate a test sample which can be used to detect the absence or the presence of a specific sequence of genetic material, a virus or even bacterium.

This technology can thus be used in the detection of infectious organisms and the detection of genetic variations including mutations.

What is PCR?
Polymerase Chain reaction (PCR) is a specific in vitro technique which allows the cloning of DNA in a test tube. This is reaction is enabled without the by-product of having to clone and subclone bacteria to do so.

For a typical PCR reaction to be performed some knowledge of the sequence information of at least two specific regions of the DNA of interest is needed so that primers can be synthesised. The primers used are designed so that they are a certain distance apart and so that they are orientated with their 3′ ends facing each other.

The process of PCR undergoes a few steps. The first step is denaturation. This is a ‘hot start’ technique which dissociates the target DNA by briefly heating the chain to 93 degrees before the DNA polymerase is added. The strands, dNTPs, primers and the DNA polymerase can then be incubated at a temperature (between 40 and 55 degrees) at which the primers hybridize onto the target strands . The DNA strands are then heated further so that elongation occurs so the first round of synthesis will occur to the DNA templates.

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In the second part of the temperature cycle the DNA products are dissociated by the denaturation step which separates the newly formed strands from the template DNA used. This separation of the newly formed strands from the DNA template can then be further passed to the next stages in the PCR reaction. In the next stage the primers now hybridize to the original stands and also the newly synthesised strands where elongation again occurs.

The third and any subsequent cycles see the amplification of the molecules between the regions encapsulated by the two primers. And after thirty cycles the increase in number of cloned molecules sees in excess of over one hundred million copies of the sequence between the primers.

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