How does a molecular beacon work?
Posted August 29, 2023
A molecular beacon works by using a stem-loop structure and holds the fluorophore and quencher in close proximity to each other. MB have covalently linked fluorescent and quenching dyes at each end of the ssDNA molecule. When free in solution, the probe is maintained in a hairpin conformation by complementary sequences at both ends of the probe. This causes FRET to occur, which suppresses reporter fluorescence. The fluorescence emission of the fluorophore is highly suppressed if there is no target. In the presence of a target sequence, the loop hybridizes to the complementary target sequence throughout the annealing step of the MB and causes the stem helix to open, at which point fluorescence is restored due to the spatial separation of the fluorophore from the quencher. The measured fluorescence signal is directly proportional to the quantity of target DNA.