Technology

Linear Probes

Linear probes are the most prevalent in assays developed thus far and function extremely well in many assay systems. They report the presence of a target DNA by first annealing to complementary target via their probe sequence. Depending on the exact type of linear probe being used, the annealing event (and therefore presence of target sequence) is 'reported' in one of two ways. For hydrolysis probes (Taqman probes), where the quencher and reporter molecules are on the same probe, the quenched fluorescent reporter molecule regains fluorescence as it is cleaved from the probe by the 5' exonuclease activity of the Taq DNA polymerase. For hybridisation probes (Lightcycler) where quencher and reporter are present on two separate probes, which only come into near contact with each other during the annealing phase of PCR, the quenching is lost, and fluorescence detected, as the two probes are melted from the target DNA by an increase in temperature.

Quenching in linear probes is obtained by Fluorescent Resonant Energy Transfer, FRET. For FRET quenching to occur, the donor molecule must be spatially close (1 - 5 bases apart) to the receptor molecule and have overlapping fluorescent spectra. As the donor molecule is excited energy is transferred to the receptor molecule and donor fluorescence is quenched. FRET based probes report when the reaction mechanism results in an increase in the spatial distance between reporter and receptor and quenching is lost.