The development of instruments that allow real-time monitoring of fluorescence within a PCR reaction vessel led to a significant advance in PCR technology and applications. Many different 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.
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 fluorimetry capabilities, real-time machines are also compatible with alternative amplification methods such as NASBA, provided a fluorescence end-point is available.