The probe consists of two types of fluorophores, which are the fluorescent parts of reporter proteins (Green Fluorescent Protein (GFP) has an often-used fluorophore). While the probe is attached or unattached to the template DNA and before the polymerase acts, the quencher (Q) fluorophore (usually a long-wavelength colored dye, such as red) reduces the fluorescence from the reporter (R) fluorophore (usually a short-wavelength colored dye, such as green). It does this by the use of Fluorescence (or Förster) Resonance Energy Transfer (FRET), which is the inhibition of one dye caused by another without emission of a proton. The reporter dye is found on the 5’ end of the probe and the quencher at the 3’ end. (www.probes.com 2003)
Figure 2. The TaqMan® probe binds to the target DNA, and the primer binds as well. Because the primer is bound, Taq polymerase can now create a complementary strand. Image created by Dan Pierce.
Once the TaqMan® probe has bound to its specific piece of the template DNA after denaturation (high temperature) and the reaction cools, the primers anneal to the DNA. Taq polymerase then adds nucleotides and removes the Taqman® probe from the template DNA. This separates the quencher from the reporter, and allows the reporter to give off its emit its energy. This is then quantified using a computer. The more times the denaturing and annealing takes place, the more opportunities there are for the Taqman® probe to bind and, in turn, the more emitted light is detected. (www.probes.com 2003)
Figure 3. The reporter dye is released from the extending double-stranded DNA created by the Taq polymerase. Away from the quenching dye, the light emitted from the reporter dye in an excited state can now be observed. Image created by Dan Pierce.
