PCR: The polymerase chain reaction
technique is a vital tool in many areas of life sciences. PCR is a method for the amplification of DNA that has revolutionised molecular biology and has many and varied applications in many and diverse areas of biological and clinical science. The PCR techniques in use today are almost exclusively based on real-time PCR, or quantitative PCR (qPCR) technology. Current real-time PCR technology has applications in a huge range of scientific areas. We provide here a review of PCR technology for competent and advanced users. Most of the information provided on these pages is from published books and papers written by leading scientists. The review includes PCR protocols, procedures and techniques, PCR troubleshooting, a description of PCR machines, tips and advice on primer design, PCR applications and much more. Also provided is a full review of PCR theory, both traditional, legacy PCR and real-time quantitative PCR or qPCR.
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The PCR blog provides a regularly updated resource on PCR technology, PCR methods, PCR applications and PCR optimization. In addition, it provides regular features on PCR troubleshooting and a bibliography for scientists and researchers who require more comprehensive information.
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PCR Theory and Technology
What is PCR?
- PCR Theory An introduction to the theory of the polymerase chain reaction
- Real-Time PCR Real-time PCR allows monitoring of the progress of a PCR reaction while the reaction is actually in progress. The technology also permits the quantitation of DNA products
- PCR Technology
- Publications on PCR
The polymerase chain reaction
(PCR) is a technique widely used in molecular biology, microbiology, genetics, diagnostics, clinical laboratories, forensic science, environmental science, food science, hereditary studies, paternity testing, and many other applications. The name, polymerase chain reaction, comes from the DNA polymerase used to amplify (replicate many times) a piece of DNA by in vitro
enzymatic replication. The original molecule or molecules of DNA are replicated by the DNA polymerase enzyme, thus doubling the number of DNA molecules. Then each of these molecules is replicated in a second "cycle" of replication, resulting in four times the number of the original molecules. Again, each of these molecules is replicated in a third cycle of replication. This process is known as a "chain reaction" in which the original DNA template is exponentially amplified. With PCR it is possible to amplify a single piece of DNA, or a very small number of pieces of DNA, over many cycles, generating millions of copies of the original DNA molecule. PCR has been extensively modified to perform a wide array of genetic manipulations, diagnostic tests, and for many other uses. For more detailed information see PCR Basic Theory ...
What is Real-Time PCR
The original or "legacy" PCR technique is performed in a tube and when the reaction is complete the products of the reaction (the amplified DNA fragments) are analysed and visualised by gel electrophoresis. Real-time PCR
permits the analysis of the products while the reaction is actually in progress. This is achieved by the use of fluorescent dyes that react with the amplified product and can be measured by a detection system
. This also permits the quantitation of the DNA. Quantitative PCR (qPCR), as this technique is known, is used to measure the quantity of a PCR product. The terms real-time PCR
are often used interchangeably although strictly speaking real-time PCR is not necessarily quantitative (though it almost always is). Similarily quantitative PCR (qPCR) does not have to be performed in real-time; there are ways of performing quantitative PCR using legacy PCR techniques. However, for practical purposes "real-time PCR" and "qPCR" may be regarded as referring to the same technology. For more detailed information about real-time PCR please see Real-Time PCR Theory ...
Real-Time PCR Machines
Real-time PCR Applications
Real-time PCR (or qPCR) is now used in almost all applications in place of traditional, legacy PCR. Real-time PCR has applications
in all branches of biological science. Applications include agricultural and food industries
, gene expression analysis
, the diagnosis of infectious disease
and human genetic testing. Due to their capability in fluorimetry the real-time machines are also compatible with alternative amplification methods such as NASBA provided a fluorescence end-point is available.
PCR Primer Design
- Real-Time PCR Applications PCR is in widespread use in diverse scientific areas.
- Real-Time PCR in forensic science The use of PCR in forensic science and forensic applications, including real-time fluorescence-based quantitative polymerase chain reaction, PCR microchip applications in forensic analysis, and PCR methods based on mitochondrial gene
Strict guidelines should be observed for optimal and accurate primer design. In any PCR or real-time qPCR procedure it is important to optimise primer design and primer concentration. For more detailed information see PCR Primer Design ...
See also: PCR Troubleshooting and Optimization
Books on real-time PCR range from basic beginners level PCR books to high-level books on PCR Troubleshooting and books aimed at advanced users on specialist PCR topics. It is difficult to choose the right book from the huge range of PCR books available. We provide excerpts from independent book reviews written by scientists in the field and published in academic journals. Read these at PCR book reviews ...
We also list our recommended PCR Books
A range of free download articles and reviews on the topic of polymerase chain reaction are available written by leading experts in the field.
A collection of PCR protocols that are available free online. The protocols are taken from published papers in peer-reviewed journals or from edited books. Available at PCR Protocols
A guide to other websites about PCR PCR Guide ...
Whether you are developing a new modification, application or variation of the PCR procedure or simply using a new source of DNA template, there is much that can go wrong. Sometimes the PCR simply doesn't work. Possible tweaks include changing the annealing time or temperature, the elongation time or temperature, changing the denaturation time or temperature, or a whole host of other variables. It may simply be a problem with the polymerase enzyme or the pH of the buffer solution. The permutations and variations are almost astronomical and you need a good logical method to overcome any PCR problems. Many scientists have found our troubleshooting guides invaluable Legacy PCR Troubleshooting Guide
and Real-Time PCR Troubleshooting and Optimization Guide
Edited by: Nick A. Saunders and Martin A. Lee read more ...
Provides both the novice and experienced user with an invaluable reference to a wide-range of real-time PCR technologies and applications and supplies detailed technical insights into the underlying principles, methods and practice of real-time PCR.
Edited by: David Rodríguez-Lázaro read more ...
An indispensable manual on real-time PCR for scientists in the food industry and for anyone involved in the detection of foodborne pathogens.
Edited by: Martin Filion"useful book ... filled with valuable information" (Doodys) read more ...
Aimed specifically at microbiologists, this volume describes and explains the most important aspects of current real-time quantitative PCR (qPCR) strategies, instrumentation and software.
Edited by: Suzanne Kennedy and Nick Oswald"an essential book ... a valuable tool to all those interested in PCR" (Doodys); "an essential guide" Aus. J. Med. Sci. read more ...
Control, optimize and troubleshoot PCR, reverse transcriptase PCR, real-time PCR and quantitative PCR. An essential book.
Edited by: Keith E. Herold and Avraham Rasooly "a comprehensive and felicitous compendium" (Drug Research) read more ...
Applications in the biomedical and life sciences: biomolecule separation, electrophoresis, chromatography, protein and cell separation, genetic and transcriptome analysis, PCR, cell viability analysis and microorganism capturing.