Figure 6-10: The Taqman^TM 5' exonuclease assay.

In addition to two conventional PCR primers, P1 and P2, which are specific for the target sequence (P1 being for instance allele specific), a third primer, P3 is designed to bind specifically to a site on the target sequence downstream of the P1 binding. P3 is labeled with two fluorophores, a reporter dye (R) is attached at the 5' end, and a quencher dye (D), which has a different emission wavelength to the reporter dye, is attached at its 3' end. Because its 3' end is blocked, primer P3 cannot by itself prime any new DNA synthesis. During the PCR reaction, Taq DNA polymerase synthesizes a new DNA strand primed by P1 and as the enzyme approaches P3, its 5'-> 3' exonuclease activity processively degrades the P3 primer from its 5' end. The end result is that the nascent DNA strand extends beyond the P3 binding site and the reporter and quencher dyes are no longer bound to the same molecule. As the reporter dye is no longer in close proximity to the quencher, the resulting increase in reporter emission intensity is easily detected.