What is PCR used for?
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, 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. See PCR Theory.
What is PCR used for?
The polymerase chain reaction is used by a wide spectrum of scientists in an ever-increasing range of scientific disciplines. In microbiology and molecular biology, for example, PCR is used in research laboratories in DNA cloning procedures, Southern blotting, DNA sequencing, recombinant DNA technology, to name but a few. In clinical microbiology laboratories PCR is invaluable for the diagnosis of microbial infections and epidemiological studies. PCR is also used in forensics laboratories and is especially useful because only a tiny amount of original DNA is required, for example, sufficient DNA can be obtained from a droplet of blood or a single hair.
Traditionally, PCR 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. However, Real-Time PCR permits the analysis of the products while the reaction is actually in progress. This is achieved by using various fluorescent dyes which react with the amplified product and can be measured by an instrument. This also facilitates the quantitation of the DNA. Not only can one tell instantly "what" DNA is present in the sample but also "how much". Quantitative PCR (Q-PCR), as this technique is known, is used to measure the quantity of a PCR product (usually in a real-time PCR procedure). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. PCR is therefore often used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Another advantage of Real-Time PCR is rapidity of the assay, since it is not necessary to perform electrophoresis or other procedure after the DNA amplification reaction.
The development of fluorescent methods for a closed tube polymerase chain reaction has greatly simplified the process of quantification. Current approaches use fluorescent probes that interact with the amplification products during the PCR to allow kinetic measurements of product accumulation. These probe methods include generic approaches to DNA quantification such as fluorescent DNA binding dyes. There are also a number of strand-specific probes that use the phenomenon of Fluorescent Resonance Energy Transfer (FRET). The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a very significant advance in PCR technology. The technology is very flexible and many alternative instruments and fluorescent probe systems are currently available. Real-time PCR assays can be completed very rapidly since no manipulations are required post-amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with size analysis on gels. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Further investigation of the real-time PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations. Real-time PCR has applications in many branches of biological science. Applications include 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.
Many books have been written on the subject of PCR. Here are a small number of 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.
Many and varied protocols are available for PCR depending on the application and procedure. A useful collection of PCR protocols available free online is available at the PCR Protocols web page.
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 the PCR Troubleshooting Guide to be useful.
Featured books: Real-Time PCR: Current Technology and Applications | Real-Time PCR in Microbiology | Microbiology Books | PCR Books | Molecular Biology Books | Epigenetics