PCR

Definition

PCR is a technique that uses the two matching strands in DNA to amplify a targeted DNA sequence from just a few samples to billions of copies within just a couple of hours.

The photo shows a scientist studying a DNA band under UV light which has been created using PCR. Credit: Pablo Rojas, Wellcome Images.

Importance

PCR has radically reduced the time and number of steps required to create large quantities of DNA for use in multiple applications. Where it has had some of its most profound impact has been in the diagnostics field. Importantly, it has increased the speed and accuracy of diagnostics. PCR is instrumental, for example, in the identification of small DNA sequences involved in cancer and genetic disorders, such as cystic fibrosis, and cancer, and for the identification and diagnosis of diseases caused by fungi, bacteria and viruses. PCR is not only used in healthcare, but is an invaluable tool for analysing ancient DNA and understanding biological evolution and for agriculture where it is used to improve the resilience and output of crops as well as animal livestock. PCR also underpins genetic fingerprinting in forensic science.

Discovery

As early as 1953, when James Watson and Francis Crick first unravelled the structure of DNA at Cambridge University, they hypothesised that one day it would be possible to copy genetic material. Three years later Arthur Kornberg, based at Washington University St Louis, identified and isolated DNA polymerase, an enzyme that replicates a cell's DNA. Following this, in the early 1960s, H. Gobind Kohrana, an Indian-American biochemist based at the Massachusetts Institute of Technology, found a means to synthesise DNA oligonucleotides, short bits of nucleic acids vital for artificial gene synthesis.

In 1971 Kjell Kleppe, a Norwegian researcher working with Kohrana, came up with the founding principle for PCR. He suggested bracketing a targeted DNA sequence using a pair of primers, strands of nucleic acids which serve as the starting point for DNA synthesis, and then copying the sequence using DNA polymerase. This idea was to become much easier to put into action with the development of techniques to sequence DNA by Fred Sanger at Cambridge University in 1977.

The first practical demonstration of PCR was achieved by Kary Mullis in 1983, who tested the technique as part of a project to understand the genetic cause of sickle cell anaemia at Cetus Corporation. Mullis, however, struggled to replicate his initial success, because the polymerase he chose for the operation kept being destroyed by the high temperatures ((95 centigrade) required to split apart (denature) the DNA strands at the start of each replication cycle. He resolved the matter by the adoption of another polymerase in 1985. This was a polymerase isolated from a species of bacterium Thermus aquaticus (Taq) discovered at a hot spring in Yellowstone National Park by Thomas D Brock in 1969. Importantly Taq polymerase proved able to withstand the high temperatures required to break apart the DNA strands at the start of the PCR cycle.

In 2000 a group of Japanese researchers led by Tsugunori Notomi published a new DNA amplification method which was much simpler and less expensive than PCR. It is called loop mediated isothermal amplification (LAMP). It has the advantage that unlike conventional PCR, it does not require the use of a thermal cycler because it amplifies DNA in a single reaction at a constant temperature. This is achieved by using a polymerase with built-in strand displacement capacities, which eliminates the need for the high-temperature denaturation step undertaken in PCR. Another point in its favour is that, unlike PCR, it does not need a separate method to analyse the product. This is done either through the use of real-time fluorimetry which enables visual detection during amplification or a post-reaction analysis using manipulation like electrophoresis. LAMP also produces considerably larger amounts of DNA than PCR, up to 50 times more, and it can be used to amplify RNA and microRNAs. Just how much of an improvement the new technique has made can be seen from the fact that LAMP was mentioned in 2000 publications between 2000 and 2017. This included article in areas all the way from infectious disease, food safety, clinical diagnostics, agricultural testing through to livestock breeding.

Application

PCR is used for a wide range of applications in science, industry, medicine, agriculture and conservation. In health and medicine it is used to advance our understanding of cancer and human genetic diseases, such as cystic fibrosis and Parkinson's. PCR is also important to the genetic identification of fungal, bacterial and viral disease. It is used, for example, to detect gonorrhoea and chlamydia in urine samples. PCR also helps determine maternity, paternity, and other blood relationships and is used by forensic scientists to identify individuals based on their genetic profile. The technique also gives scientists the means to hunt for similar genes scattered throughout different species or populations to determine the evolutionary distance of related species and to amplify ancient DNA isolated from samples of extinct animals. PCR is also vital to conservation work, helping to prevent the inbreeding of captive animals and to identify the origin of illegally obtained ivory. The technique is also used in agriculture to determine plant species, pests and diseases.

One of the limitations with PCR is that it requires equipment and infrastructure that are difficult to shift around with the user. Some of these problems are being overcome by the adoption of the LAMP technique which is easier to carry out in the field because it needs less complex and less expensive instrumentation. It also has the advantage that it can amplify DNA from partially processed and/or non processed samples and is extremely fast.

LAMP is now a widely used molecular diagnostic method and stands at the forefront of the point-of-care diagnostic revolution. Due to its simplicity, low cost, low-energy consumption, ease of learning and robustness, LAMP is proving a useful diagnostic and detection technique for screening for infectious diseases in low and middle-income countries. Since 2005 it has begun to be used in screening programmes for malaria, tuberculosis and sleeping sickness. The method was also recently used to detect the Ebola virus. Trials are now underway to see if LAMP could provide a tool for the rapid detection of meningitis, the speedy treatment of which is currently hampered by the difficulty in identifying the disease before infection overwhelms the body.

PCR: timeline of key events

An American biochemist, Mullen was jointly awarded 1993 Nobel Prize in Chemistry for development of polymerase chain reaction (PCR) method to quickly make a huge number of copies of specific pieces of DNA.1944-12-28T00:00:00+0000Kjell Kleppe, a Norwegian scientist working in H. Gobind Khorana's Institute for Enzyme Research at University of Wisconsin publishes papers describing the principles of PCR.1969-01-01T00:00:00+0000Called Thermus aquaticus (Taq) this enzyme becomes a standard source of enzymes because it can withstand higher temperatures than those from E Coli. Taq is later important in the PCR technique. 1969-01-01T00:00:00+0000K. Kleppe, E Ohtsuka, R Kleppe, I Molineux, HG Khorana, "Studies on polynucleotides *1, *2XCVI. Repair replication of short synthetic DNA's as catalyzed by DNA polymerases", Journal of Molecular Biology, 56/2 (1971), 341-61. The method provides an artificial system of primers and templates that allows DNA polymerase to copy segments of the gene being synthesised. 1971-01-01T00:00:00+0000Kary Mullis, an American biochemist based at Cetus, proposed an alternative method to Sanger's DNA sequencing method to analyse Sickle cell Anaemia mutation which laid the foundation for the development of the PCR technique. 1983-05-01T00:00:00+0000Mullis reports on his production of olgionucleotides and some results from his experiments with PCR to Cetus Corporation's annual meeting but few show any interest. 1984-06-01T00:00:00+00001985-01-01T00:00:00+0000The application establishes polymerase chain reaction (PCR) as a method for amplifying DNA in vitro. PCR uses heat and enzymes to make unlimited copies of genes and gene fragments. The application is broad and is based on analysis of Sickle Cell Anaemia mutation via PCR and Oligomer restriction. 1985-03-01T00:00:00+00001985-12-01T00:00:00+0000The technique enabled the amplification of small fragments of DNA on a large scale. It was published in RK Saiki et al, Enzymatic Amplification of beta-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia, Science, 230 (1985), 1350-54.1985-12-20T00:00:00+0000Gregory Winter at the Laboratory of Molecular Biology develops the technique as part of his strategy to create an artificial immune system for generating monoclonal antibodies. The technique is published in R Olandi, DH, Gussow, PT Jones and G Winter, 'Cloning immunoglobulin variable domains for expression by polymerase chain reaction', Proc Natl Acad Sci USA, 86 (May 1989), 3833-7. 1988-11-01T00:00:00+00001989-01-01T00:00:00+0000Cetus was the first biotechnology company to be founded1993-10-13T00:00:00+0000T. Nogtomi, H. Okayama, H. Masubuchi, T. Yonekawa, K. Wantabe, N. Amino, T. Hase, 'Loop-mediated isothermal amplification', Nucleic Acids Research, 28/12 (2000), e63.2000-06-15T00:00:00+0000
Date Event People Places
December 1944Kary Banks Mullis was bornCetus Corporation
1969First principles for PCR publishedKhorana, KleppeUniversity of Wisconsin-Madison
1969New species of bacterium is isolated from hot spring in Yellowstone National Park by Thomas BrockBrockCase Western Reserve University
1971Process called repair replication for synthesising short DNA duplexes and single-stranded DNA by polymerases is publishedKhorana, KleppeMIT
1983Polymerase chain reaction (PCR) starts to be developed as a technique to amplify DNAMullisCetus Corporation
June 1984Results from PCR experiments start being reportedMullisCetus Corporation
January 1985 - May 1987Cetus developed PCR for measuring amount of HIV circulating in bloodCetus Corporation
March 1985Mullis and Cetus Corporation filed patent for the PCR techniqueMullisCetus Corporation
December 1985Cetus Corporation and Perkin-Elmer partnered to develop instruments and reagents for PCRCetus Corporation
20 Dec 1985The Polymerase Chain Reaction technique was publishedMullisCetus Corporation
November 1988Patent application filed for the the use of PCR to create a library of antibody fragmentsGussow, Jones, Olandi, WinterLaboratory of Molecular Biology, Istituto Nazionale dei Tumori
January 1989PCR technique starts being used for DNA finger printing tests 
October 1993Cetus Corporation was sold to Chiron and its patent rights sold for US$300 million to Hoffman-La RocheCetus Corporation, Chiron, Hoffman-La Roche
15 Jun 2000New simpler and cheaper PCR method published opening possibility for use in middle to low-income countriesNogtomi, Okayama, Masubuchi, Yonekawa, Wantabe, AminoUniversity of Tokyo, Osaka University, Eiken Chemical Co

Dec 1944

Kary Banks Mullis was born

1969

First principles for PCR published

1969

New species of bacterium is isolated from hot spring in Yellowstone National Park by Thomas Brock

1971

Process called repair replication for synthesising short DNA duplexes and single-stranded DNA by polymerases is published

1983

Polymerase chain reaction (PCR) starts to be developed as a technique to amplify DNA

Jun 1984

Results from PCR experiments start being reported

Jan 1985 - May 1987

Cetus developed PCR for measuring amount of HIV circulating in blood

Mar 1985

Mullis and Cetus Corporation filed patent for the PCR technique

Dec 1985

Cetus Corporation and Perkin-Elmer partnered to develop instruments and reagents for PCR

20 Dec 1985

The Polymerase Chain Reaction technique was published

Nov 1988

Patent application filed for the the use of PCR to create a library of antibody fragments

Jan 1989

PCR technique starts being used for DNA finger printing tests

Oct 1993

Cetus Corporation was sold to Chiron and its patent rights sold for US$300 million to Hoffman-La Roche

15 Jun 2000

New simpler and cheaper PCR method published opening possibility for use in middle to low-income countries