from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

Real-time PCR requires monitoring the reaction during amplification. Fluorescence is a convenient method of interrogation that only requires a clear optical path for excitation and emission. Double-stranded DNA (dsDNA) dyes and fluorescently-labeled probes are both commonly used. dsDNA dyes directly measure the amount of double-stranded product produced. Probes used in real-time PCR function indirectly through fluorescence resonance energy transfer (FRET) or fluorescence quenching. Initially proposed in the late 1940s, it was not until the 1980s that FRET was applied to DNA (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). However, real-time monitoring with probes was only achieved several years later after dsDNA dyes were established in real-time PCR. One advantage of probes over dsDNA dyes is multiplexing by color with different fluorescent dyes. Nevertheless, this advantage comes at a cost in instrumentation and analysis complexity. Furthermore, multiplex analysis with dsDNA dyes is possible by melting temperature separation of products and/or probes.