What is the principle of the 5' nuclease assay (TaqMan)?
A) Fluorescent probe is mixed with labeled primers and a sandwich occurs where the fluorescent molecule is activated to fluorescence.
B) The Taq DNA polymerase extends from the primers and replicates the template to which the TaqMan probe is annealed. The reporter dye is released by the 5' nuclease activity of the polymerase first and then the probe is released, thus increasing the fluorescence.
C) The Taq DNA polymerase extends from the primers and replicates the template to which the TaqMan probe is annealed. Then the reporter dye is activated and the fluorescence increases with the number of hybrids that are produced.
D) A bioluminescent probe is attached to the 5' side of the primer. As the Taq DNA polymerase begins adding nucleotides to the growing chain, the last nucleotide added (because of steric hindrance) will be the bioluminescent probe. Once added, the probe will begin luminescing and the concentration of the hybrids is directly proportional to the amount of luminescence that is produced.

Question

Quizplus · Accepted Answer

The TaqMan assay involves a fluorescent probe that is designed to anneal to a specific sequence within the target DNA. This probe contains a fluorescent dye and a quencher molecule, which prevents the dye from fluorescing. As the Taq DNA polymerase extends from the primers and replicates the template, it encounters the probe, which undergoes 5' nuclease activity by the polymerase. This releases the reporter dye from the probe, allowing it to fluoresce and increase the overall fluorescence signal. Therefore, the principle of the TaqMan assay is based on 5' nuclease activity of the polymerase and the subsequent release of the reporter dye from the probe.

What Is the Principle of the 5' Nuclease Assay (TaqMan)