During PCR the probe will bind to a specific site which contains its complementary sequence. The Amplitaq Gold Polymerase will then come and cleave the probe, such that the fluorescence molecule separates from the quencher molecule. This will then allow fluorescence to be detected using the 7700 Sequencer. If, on the other hand, the complementary strand containing the polymorphism does not exist in the DNA sample, then the probe will not bind. Thus, when the Amplitaq Gold Polymerase replicates the DNA sample, the probe will remain intact with the quencher molecule absorbing the fluorescence.
Several problems arise, however.
Because the probe contains both a fluorescence and a quencher molecule, the overall price of the probe increases dramatically. Therefore, any large-scale use of the TaqMan assay is rather expensive. In addition, the ability to multiplex is limited. FP is based on the observation that when a fluorescent molecule is excited by plane-polarized light, it emits polarized fluorescent light into a fixed plane if the molecules remain stationary between excitation and emission. Because the molecule rotates and tumbles in space, FP is not fully detected by an external detector.