The Polymerase chain reaction (PCR) is a genetic technique that occurs in vitro to allow the specific enzymatic amplication of specific DNA fragments of certain sequence and length. This revolutionary technique uses a template of DNA, oligonucleotide primers and the thermostable enzyme Taq polymerase, to reproduce many copies of the initial molecule.
1971 brought the publication of an initiative[1] that proposed using two primers in a repair replication reaction allowing two new strands of duplex DNA to be synthesised from a double stranded duplex. This technique didn’t become widely used until the publication of PCR in 1987[2] after which certain advances in the PCR technology soon made it the method of choice for scientists involved in gene cloning and analysis.
In PCR, knowledge of sequences that flank the region to be amplified is required. By automated chemical synthesis oligonucleotides complementary to these sequences are produced and are used as primers in a series of DNA polymerase-catalysed reactions. Using a thermostable form of DNA polymerase (Taq polymerase)which does not become denatured at high temperatures avoids the need to include additional polymerase at each cycle. The cycle is repeated 30 or more times, with each cycle increasing the amount of duplex DNA molecules present.
Since then there have been major innovations made, as applications of PCR have been essential to the progression of certain areas in science. These include the mapping of the human genome project, single sperm analysis, molecular archaeology and ancient DNA, molecular ecology and behaviour, disease diagnosis and drug discovery.
PCR has enabled the analysis of molecules from the past in order to examine species that may now be extinct and to determine their relationships to present day organisms. One example of this analysis is, DNA from a 5000 year old frozen mummy, the Tyrolean ice man, was analysed in a hypervariable region of mitochondrial genome and this late Neolithic individual’s mitochondrial type was found in 13 of 1200 people and was closely related to the types determined from central and northern Europe populations.
Within ecology, PCR plays an important role in integrating genetic data with field observations of various species to address topics such as mating systems, hybrid zones, sex identification and many more. As PCR can obtain multilocus genotypes non-invasively it has become the method of choice to determine genetic relationships between large organisms.
PCR helped study a variety of chronic virus infections (HIV, HCV, hepatitis B virus, human papillomavirus, cytomegalovirus). PCR has been crucial for the detection of HIV infection in neonates, Quantitation of HIV and HCV viremia by PCR has been used to monitor response to drug therapy. PCR is also useful for the rapid diagnosis of pulmonary infections in immuno-compromised hosts, like for cytomegalovirus
Two significant areas of PCR application, the diagnosis of infectious diseases and genetic testing are being developed further by the introduction of real time, kinetic, homogeneous PCR reactions and also microarray techniques. These continuing developments will increase the already major role of PCR in areas of clinical and social relevance, including prognostic evaluation of disease states and drug discovery and blood screening.
[1] Kleppe, K. et al. (1971) J.mol.Biol. 56, 341-361
[2] Mullis, K.B. and Foloona, F.A(1987). Methods enzymol. 155, 335-350
