TaqMan probes consist of a fluorophore
covalently attached to the 5’-end of the
oligonucleotide probe and a quencher at the 3’-end. Several different fluorophores (e.g.
6-carboxyfluorescein, acronym:
FAM, or tetrachlorofluorescein, acronym: TET) and
quenchers (e.g. tetramethyl
rhodamine, acronym: TAMRA) are available. The quencher molecule quenches the
fluorescence emitted by the fluorophore when excited by the cycler’s light source via
Förster resonance energy transfer (FRET). As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals. TaqMan probes are designed such that they anneal within a DNA region amplified by a specific set of primers. (Unlike the diagram, the probe binds to single stranded DNA.) TaqMan probes can be conjugated to a minor groove binder (MGB) moiety, dihydrocyclopyrroloindole tripeptide (DPI3), in order to increase its binding affinity to the target sequence; MGB-conjugated probes have a higher melting temperature (Tm) due to increased stabilization of van der Waals forces. As the
Taq polymerase extends the
primer and synthesizes the nascent strand (from the single-stranded template), the 5' to 3'
exonuclease activity of the
Taq polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the
quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR. ==Applications==