Real time PCR

Abstract

Polymerase Chain Reaction (PCR) has revolutionised molecular biology, and is now a well-standardised procedure and its applications are wide. Real time PCR is a more recent development of the initial PCR reaction, which has provided a solution to the limitation of standard PCR with regards to quantification. By using fluorescence, which allows the quantification of PCR products at each cycle of the reaction, real time PCR avoids the "plateau" effect of standard PCR. Two main approaches have been developed, the TaqMAn assay and the SYBR-Green I dye. Alongside real-time PCR as such, an "end point assay" has also evolved, most often referred to as allelic discrimination, allowing the detection of a specific sequence (i.e. genetic polymorphism). The technology is now widely used for DNA and RNA quantification as well as genetic polymorphism detection. It can be applied to the detection and quantification of plain mRNA, for splice variant, alternative promoter usage, allele specific expression etc. For DNA, gene amplification, gene deletion or rearrangement, allelic copy number can be measured. It is also widely used for the detection of pathogens or contaminants in food or other types of products.