An Introduction to Real-Time PCR

The development of instruments that allowed real-time monitoring of fl uorescence within PCR reaction vessels was a very signifi cant advance. The technology is very flexible and many alternative instruments and fl uorescent probe systems have been developed and are currently available. Real-time PCR assays can be completed very rapidly since no manipulations are required post-amplifi cation. Identifi cation of the amplifi cation products by probe detection in realtime is highly accurate compared with size analysis on gels. Analysis of the progress of the reaction allows accurate quantifi cation 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 fi rst 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. caister.com/pcrbooks