Polymerase chain reaction

The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.^[1]

PCR is fundamental to many of the procedures used in genetic testing and research, including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory research for a broad variety of applications including biomedical research and forensic science.^[2][3]

The majority of PCR methods rely on thermal cycling. Thermal cycling exposes reagents to repeated cycles of heating and cooling to permit different temperature-dependent reactions—specifically, DNA melting and enzyme-driven DNA replication. PCR employs two main reagents—primers (which are short single strand DNA fragments known as oligonucleotides that are a complementary sequence to the target DNA region) and a thermostable DNA polymerase. In the first step of PCR, the two strands of the DNA double helix are physically separated at a high temperature in a process called nucleic acid denaturation. In the second step, the temperature is lowered and the primers bind to the complementary sequences of DNA. The two DNA strands then become templates for DNA polymerase to enzymatically assemble a new DNA strand from free nucleotides, the building blocks of DNA. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the original DNA template is exponentially amplified.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. If the polymerase used was heat-susceptible, it would denature under the high temperatures of the denaturation step. Before the use of Taq polymerase, DNA polymerase had to be manually added every cycle, which was a tedious and costly process.^[4]

Applications of the technique include DNA cloning for sequencing, gene cloning and manipulation, gene mutagenesis; construction of DNA-based phylogenies, or functional analysis of genes; diagnosis and monitoring of genetic disorders; amplification of ancient DNA;^[5] analysis of genetic fingerprints for DNA profiling (for example, in forensic science and parentage testing); and detection of pathogens in nucleic acid tests for the diagnosis of infectious diseases.