The RT step synthesizes a cDNA copy of the RNA template. After denaturation,primers and probe anneal to their targets. The probe contains a reporter dye at the 5 end and a quencher (Q) at its 3 end. During the polymerization step, the 5 nuclease activity of the Taq polymerase displaces and cleaves the probe. This physically separates the reporter dye and quencher dyes, resulting in reporter fluorescence. The increase in signal is directly proportional to the number of molecules released during that cycle. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye.