Real-time PCR Definition
Real-Time PCR is a variation of the polymerase chain reaction (PCR) that allows simultaneous (i.e. in real-time) amplification and detection of DNA templates. Because it is used to quantitate DNA, it is often abbreviated to qPCR, although that abbreviation is not universally accepted.
Quantification is based on the detection of a fluorescent reporter molecule that increases as PCR product accumulates with each cycle of amplification. There are three main chemistries in general use:
1. Intercalating dyes, such as SYBR-Green, which fluoresces upon light excitation when bound to double stranded DNA. These are cheap, easily added to legacy assays and amplification products can be verified by the use of melt curves. On the other hand, they can lack specificity and fluorescence varies with amplicon length.
2. Primers linked to fluorophores, e.g. Lux primers or Plexor. Fluorescence detection depends on the primers binding to, and being extended during the PCR reaction. These are cheap and amplification products can be verified by melt curves. However, specificity depends on the primers and specific, usually company-specific design software needs to be used for optimal performance.
3. Hybridisation-probe based methods, e.g. TaqMan or Molecular Beacons. These are the most specific methods, as products are only detected if the probes hybridise to the appropriate amplification products. There are many variations on this theme, with melt curve analysis possible for some chemistries. Their main disadvantages are cost, complexity and occasional fragility of probe synthesis and potential problems associated with the fact that probe-based assays do not report primer dimers, that can interfere with the efficiency of the amplification reaction.
If real-time PCR is preceded by a reverse transcription (RT) step to quantitate mRNA or viral RNA targets, this method is often termed qRT-PCR or, alternatively, RT-qPCR.