This process of denaturing, annealing and elongation is repeated multiple (30-40) times, thereby increasing exponentially the number of copies of the desired gene in the mixture. Although this process would be quite tedious if performed manually, samples can be prepared and incubated in a programmable Thermocycler, now commonplace in most molecular laboratories, and a complete PCR reaction can be performed in 3-4 hours. Each denaturing step stops the elongation process of the previous cycle, thus truncating the new strand of DNA and keeping it to approximately the size of the desired gene. The duration of the elongation cycle can be made longer or shorter depending on the size of the gene of interest, but eventually, through repeated cycles of PCR, the majority of templates will be restricted to the size of the gene of interest alone, as they will have been generated from products of both of the primers.
A number of factors will affect whether or not a PCR is successful. Some of these are the quality of the original DNA sample, quantity of template DNA and suitability of the primers. The most widely used method to test for PCR product is gel electrophoresis which is used to separate DNA fragments based on size and charge. The fragments are then visualized using dyes or radioisotopes.
The Evolution of PCR
Since the discovery of PCR, DNA polymerases other than the original Taq have been discovered. Some of these have better “proof-reading” ability or are more stable at higher temperatures, thus improving the specificity of PCR and reducing errors from insertion of the incorrect dNTP.
Some variations of PCR have been designed for specific applications and are now used regularly in molecular genetic laboratories. Some of these are Real-Time PCR and Reverse-Transcriptase PCR. The discovery of PCR has also lead to the development of DNA sequencing, DNA fingerprinting and other molecular techniques.
