Practice question

Molecular beacons are hairpin-shaped probes bearing a fluorophore at one end and a quencher at the other. In free solution, the stem keeps them close, enabling FRET quenching with negligible emission. Hybridization occurs specifically during the annealing phase, when temperature permits complementary base pairing to target amplicons. Binding forces stem separation, physically distancing fluorophore from quencher and restoring fluorescence. Fluorescence intensity therefore reflects target-specific duplex formation at annealing, not denaturation or extension. This reversible, hybridization-dependent signaling enables real-time monitoring and enhanced specificity for SNP discrimination.