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Principle of Polymerase Chain
Reaction
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Principle of PCR
The principle of the polymerase chain reaction (PCR) is the repeated copying of a chosen segment of DNA using
specific sense and anti-sense primers, usually separated by 200-400 nucleotides on the genome or nucleic acid of
interest. With the availability of thermostable DNA polymerases derived from thermophilic bacteria this repetitive
copying of the DNA can be done in a single tube by repeatedly heating the DNA to high temperature (94°C) to
dissociate the DNA duplex, cooling to allow annealing of the primers to the template (37-60°C, depending on the
primers used) and finally heating to the optimum temperature (72°C) for the polymerase to copy the template to
produce a new DNA strand. The cycles are repeated 25-35 times (25 cycles theoretically increases the concentration
of starting template DNA 107 times) to produce a DNA product which can be directly visualised by ethidium bromide
staining on an agarose gel. The size of the DNA product is exactly defined by the location of the two primers on
the virus genome. Non-specific DNA products can sometimes be produced, but these are usually of a different size
and care is needed in interpretation of results. PCR is particularly useful in diagnosis because it is very
sensitive and highly specific, being able to distinguish between even closely related strains of pathogens.
The genome of FMD virus consists of a single strand of RNA and, since it is not DNA, it cannot be reproduced directly by PCR but must first be copied into DNA, by reverse transcription, and then amplified in a two-step reaction known as reverse-transcription/(PCR) (RT-PCR).
An advantage that RT-PCR has over DNA probe analysis is that the resulting DNA product can be sequenced, either after cloning in a suitable vector or by direct DNA sequencing of the PCR product, and from these data the relationship of the virus to other isolates can be determined. This greatly facilitates the study of the epidemiology of the disease.
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