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, 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

Fluorescence from the reporter dye is kept to a low background level because of the close proximity of the quencher dye by fluorescence resonance energy transfer (FRET),where the fluorophore donates energy to the quencher dye.During the primer extension,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 entire probe is released.Fluorescence increases as the fluorophore is removed from the immediate vicinity of the quencher dye.

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