Perhaps it was not the most friendly introduction to scientific techniques, but PCR has been all over the news for what now looks like an eternity. But PCR is one of the many lab techniques used to test for the presence or absence of gene or gene mutation. Or to check if the modifications to a particular DNA fragment are correct. For PCR to exist two major breakthroughs were needed, the first one, was the design or discovery of a stable polymerase, and the development of a device capable to change the temperature of the sample.
In both cases, temperature plays a major role in the development, and the main reason is the stability behand DNA and its dependence on temperature. When temperature increases the pairing in the different bases in the DNA chain starts to break apart. But in the cell, this unwinding of both chains is performed by a single enzyme without changes in temperature. For this reason, the different enzymes involved in the replication of DNA cannot withstand high temperatures.
But when the polymerase from a thermophilic bacterium was isolated the ability to copy or replicate DNA was within reach. As only a few reagents were needed to replicate DNA, the DNA sample, the different nucleotides in DNA, the polymerase, and small DNA fragments to direct or prime the reaction(primers).
First, the temperature is increased to separate the two strands of the DNA sample. Then by lowering the temperature the DNA starts to bind or anneal to the primers. And finally, the temperature is lowered again for the polymerase to start to extend the different chains. This cycle will return two copies of DNA and by this simple expansion, the content of DNA will increase exponentially.
By being able to direct the reaction we can test for specific fragments of DNA inside a sample. This results in the specificity of the assay. Products of PCR are then observed in an agarose gel, if there’s a fragment of an already known size, then the assay is positive, otherwise negative. In this particular format, the PCR reaction is known as endpoint PCR.
The development of new detection methods yielded a new kind of PCR. If the amount of product is detected as its generated, then the PCR is known as qPCR or quantitative PCR. In this particular configuration, the presence of a particular fragment of DNA can be tested. And also the amount of this product. With this method, the product is already detected and quantified at the end of the reaction. Removing the need to perform electrophoresis of the samples, and getting the results faster.
Quantification of the original sample in the case of a viral infection correlates with the number of viral particles in the sample. But infectivity of those particles is something that PCR cannot answer. Although these are not the only kinds of PCR, those two contain the main principles and reagents to perform the technique. See you in the next one.