Fig 4.

Principle of the TaqMan PCR. In a first step (I) the probe labelled with a fluorescing marker, R, and a quencher, Q, binds to a stretch between the two primers. As long as R is covalently bound to Q, illumination by UV does not result in fluorescence. During extension (II), the 5′-3′ exonuclease activity results in hydrolysis of the probe which leads to liberation of R (III). The amount of R released during PCR can be measured directly during the entire amplification process. The amount of fluorescence that accumulates in a tube is proportional to the amount of amplified DNA. Our example shows the FCoV TaqMan principle. FCoV1229r—first primer, FCoV1128f—second primer, FCoV1200p—probe, Tfl—heat stable Thermus flavus polymerase. For details see text and Gut et al 1999.