The principle

• RealFast™ Assays contain hydrolysis probes, also commonly called TaqMan^® probes.
• The sequence-specific oligonucleotide probes carry a fluorescent reporter dye at the 5´-end and a quencher dye at the 3´-end.
• While the probe is intact, the quencher is close enough to the reporter to suppress the fluorescent signal of the 5´-fluorophore.
• During the continuous annealing/extension phase of PCR, the probe is cleaved by the 5´ to 3´ exonuclease activity of Taq DNA polymerase, thereby spatially separating the fluorophore from the quencher dye.
• This process results in detectable fluorescence, which is proportional to the amount of accumulated PCR product.