Real-time PCR

Real-time PCR and RT-PCR (also known as quantitative or qPCR) allow accurate quantification of starting amounts of DNA, cDNA, and RNA targets. Fluorescence is measured during each cycle, which greatly increases the dynamic range of the reaction, since the amount of fluorescence is proportional to the amount of PCR product. PCR products can be detected using either fluorescent dyes that bind to double-stranded DNA or fluorescently labeled sequence-specific probes.

The fluorescent dye SYBR Green I binds all double-stranded DNA molecules, emitting a fluorescent signal of a defined wavelength on binding (see figure SYBR Green principle). The excitation and emission maxima of SYBR Green I are at 494 nm and 521 nm, respectively, allowing use of the dye with any real-time cycler. Detection takes place in the extension step of real-time PCR. Signal intensity increases with increasing cycle number due to the accumulation of PCR product. Use of SYBR Green enables analysis of many different targets without having to synthesize target-specific labeled probes. However, nonspecific PCR products and primer–dimers will also contribute to the fluorescent signal. Therefore, high PCR specificity is required when using SYBR Green.

Fluorescently labeled probes provide a highly sensitive method of detection, as only the desired PCR product is detected. However, PCR specificity is also important when using sequence-specific probes. Amplification artifacts such as nonspecific PCR products and primer–dimers may also be produced, which can result in reduced yields of the desired PCR product. Competition between the specific product and reaction artifacts for reaction components can compromise assay sensitivity and efficiency. The following probe chemistries are frequently used.

TaqMan probes: sequence-specific oligonucleotide probes carrying a fluorophore and a quencher moiety. The fluorophore is attached at the 5' end of the probe and the quencher moiety is located at the 3' end. During the combined annealing/extension phase of PCR, the probe is cleaved by the 5'–3' exonuclease activity of Taq DNA polymerase, separating the fluorophore and the quencher moiety. This results in detectable fluorescence that is proportional to the amount of accumulated PCR product.

FRET probes: PCR with fluorescence resonance energy transfer (FRET) probes uses 2 labeled oligonucleotide probes that bind to the PCR product in a head-to-tail fashion. When the 2 probes bind, their fluorophores come into close proximity, allowing energy transfer from a donor fluorophore to an acceptor fluorophore. Therefore, fluorescence is detected during the annealing phase of PCR and is proportional to the amount of PCR product. As the FRET system uses 2 primers and 2 probes, good design of the primers and probes is critical for successful results.

Dyes used for fluorogenic probes in real-time PCR: For real-time PCR with sequence-specific probes, various fluorescent dyes are available, each with its own excitation and emission maxima (see table Dyes commonly used for quantitative, real-time PCR. The wide variety of dyes makes multiplex, real-time PCR possible (detection of 2 or more amplicons in the same reaction), provided the dyes are compatible with the excitation and detection capabilities of the real-time cycler used, and the emission spectra of the chosen dyes are sufficiently distinct from one another. Therefore, when carrying out multiplex PCR, it is best practice to use dyes with the widest channel separation possible to avoid any potential signal crosstalk.

Other probes: Many probe suppliers have developed their own proprietary dyes. For further information, please refer to the web pages of the respective suppliers.

Dyes commonly used for quantitative, real-time PCR