An Introduction to Real-Time PCR
N. A. Saunders
from: Real-Time PCR: Current Technology and Applications (Edited by: Julie Logan, Kirstin Edwards and Nick Saunders). Caister Academic Press, U.K. (2009)
Abstract
The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a significant advance. The technology is flexible and many alternative instruments and fluorescent probe systems have been developed and are currently available. Real-time PCR assays can be completed 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. Since the first practical demonstration of the concept real-time PCR has found applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. Due to their fluorimetry capabilities, these real-time machines are also compatible with alternative amplification methods such as NASBA, provided a fluorescence end-point is available read more ...
