PCR Technology

Understand How the PCR Process Works

The multi-step PCR testing and analysis process begins with the sampling, which can be achieved utilizing standard field sampling techniques (i.e. swab, tape-lift, Air-O-Cell, 37mm PCM cassette filter).

Next, the sample is processed and DNA is extracted from the samples. This extracted DNA is then added to a PCR cocktail or "master mix". This master mix includes the primers specifically designed for the fungal species to be identified and enumerated.

It is then placed into a thermocycler for amplification. The first stage of the PCR reaction is the denaturation or melting of the DNA (the double helix splits apart). This is achieved by heating the sample to 95 degrees Celsius.

Next, the mixture is cooled to 45-60 degrees Celsius. This is specific to the primer sets and the annealing stage occurs. The two single stranded primers find their complementary DNA sequences and hybridize or bond to them.

The third stage of PCR amplification is extension or synthesis. The thermostable polymerase (Taq) catalyzes the addition of complementary bases to the single stranded template DNA. This forms two new double-stranded DNA helix. These three stages of PCR amplification are then repeated over and over (cycles up to 40 times) again. Hence, the Chain reaction.

PCR testing has become commonplace in research facilities across the world. In the 1990’s the commercial release of quantitative PCR (Q-PCR) marked the beginning of a new chapter in rapid molecular diagnostics. Q-PCR, commonly referred to as Real time PCR, has come to the forefront of the diagnostic and research world by providing accurate quantification of DNA in real time, while decreasing the time necessary to produce detectable levels of PCR product. With this, researchers began to apply this technology to rapid molecular level microorganism and genetic modification detection systems.

Microbac's molecular scientists routinely perform the following PCR analyses:

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