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PCR: The polymerase chain reaction

The PCR 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.

The PCR Blog

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

• PCR Theory An introduction to the theory of the polymerase chain reaction
• What is PCR? A definition of PCR
• Legacy PCR Theory The theory of traditional, legacy PCR
• Real-time PCR Theory The theory of real-time PCR
• The Principle of PCR Detailed PCR principles with equations for calculating the final amount of DNA and the final number of copies of the target sequence
• Multiplex PCR
• 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
• Theory of Real-Time PCR An introduction to the theory of real-time PCR
• Real-Time PCR Machines
• Features of Real-Time PCR Machines A comparison of different, commercially-available, machines
• Temperature Specifications Temperature specifications of real-time machines
• Fluorescence Detection Real-time PCR chemistries and detection formats
• Real-Time PCR Design
• Quantification Analysis Real-time PCR Quantification Analysis
• Real-time PCR Quality Control
• Real-time PCR Parameters
• PCR Technology
• PCR Primer Design A very important aspect of PCR and real-time PCR
• PCR Protocols PCR protocols available free online
• PCR Applications A description of some common applications of PCR and real-time PCR
• PCR in Forensic Science
• Real-Time PCR for the Detection of Foodborne Pathogens
• PCR Troubleshooting A guide to troubleshooting and optimization of PCR methods and technology
• Primer Concentration
• Template DNA
• Taq Concentration
• Inadequate dNTPs
• Magnesium Concentration
• Potassium Chloride Concentration
• Annealing Time and Temperature
• Publications on PCR
• PCR Book Reviews Excerpts from published reviews of current books on PCR
• PCR Reviews Free articles to download
• PCR Resources Online resources

What is 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 and qPCR 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
• Theory of Real-Time PCR An introduction to the theory of real-time PCR
• Real-Time PCR Machines What they do and how they work

Real-Time PCR Machines

• Theory of Real-Time PCR Machines An introduction to real-time PCR machines and an explanation of how they work
• Comparison of Real-Time PCR Machines Compare the pros and cons of various commercial PCR machines. A guide to choosing the most appropriate thermocycler for your PCR needs. How to select the best PCR machines and instruments suitable for the needs of a particular laboratory
• Temperature Specifications of qPCR machines Technical details
• Real-time PCR Chemistries and Detection Formats A review of real-time PCR chemistries and detection formats including nonspecific detection formats, hydrolysis probes, and hybridization probe

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.

• 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

PCR Primer Design

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

PCR Books

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.

PCR Articles

A range of free download articles and reviews on the topic of polymerase chain reaction are available written by leading experts in the field.

• PCR Papers

PCR Protocols

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.

PCR Guide

A guide to other websites about PCR PCR Guide ...

PCR Troubleshooting

• PCR Troubleshooting
• Legacy PCR Troubleshooting Book
• Real-Time PCR Troubleshooting and Optimization Book

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

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