(b). B, Molecular beacon. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore thai fluoresces once the beacon probe binds to the amplified target and the quencher is no longer in close proximity to the fluorophore. These probes are designed such that the loop portion of the molecule is a sequence complementary to the target of interest (a). The "stem" portion of the beacon probe is formed by the annealing of complementary aim sequences on the respective ends of the probe sequence. In addition, a fluorescent moiety (R) and a quencher moiety (Q) at opposing ends of the probe are attached (a). The stem portion of the probe keeps the fluorescent and quencher moieties in close proximity to one another such that the fluorescence of the fluorophore remains quenched. When it encounters a target molecule with a complementary sequence, the molecular beacon undergoes a spontaneous conformational change that forces the stem apart, thereby causing the fluorophore and quencher to move away from each other and leading to restoration of fluorescence (b). C, Fluorescent resonant energy transfer (FRET) or hybridization probes. Two different hybridization probes are used, one carrying a fluorescent reporter moiety at its 3' end (designated Rl) and the other probe carrying a fluorescent dye at its 5' end (designated R2) (a). These two oligonucleotide probes are designed such that they hybridize to amplified DNA target in a head-to-tail arrangement in very close proximity to one another. The fust dye (Rl) is excited by a filtered light source and emits a fluorescent light at a slightly longer wavelength. Because the two dyes are in close proximity to each other, the energy emitted from Rl excites R2 attached to the second hybridization probe, which emits fluorescent light at an even longer wavelength (b). This energy transfer is referred to as FRET. By selecting an appropriate detection channel on the instrument, the intensity of light emitted from R2 is filtered and measured. (Modified with permission from Mocellin S, Rossi CR, Pilar! P, et al: Quantitative real-time PCR: a powerful ally in cancer research. Trends Mol Med 9:189,2003.)
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