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.
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