Practice question

TaqMan chemistry exploits the 5' to 3' exonuclease activity of Taq polymerase. The linear dual-labeled probe hybridizes to template during annealing, and during extension polymerase cleaves the probe, separating fluorophore from quencher and producing irreversible fluorescence accumulation. Molecular beacons instead rely on reversible hybridization without cleavage; their stem-loop opens at annealing when bound to target and closes after denaturation, yielding transient fluorescence at annealing phase. TaqMan does not possess stem-loop structure, nor does it bind minor grooves. The key mechanistic difference is fluorescence generation during extension via hydrolysis versus conformational opening at annealing.