30 May 2011

PCR Real-Time PCR

Usually, PCR is performing 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...

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