Molecular beacons are stem-loop (hairpin) shaped hybridization probes with a fluorescent dye and a quencher dye on the opposite extremities brought to their proximity by the complementary stem (Fig. 18.2c). The commonly used fluorescent dyes are FAM, TAMRA, TET, and ROX paired with a quenching dye, typically DABCYL. While in the absence of amplicon target, the FRET between the fluorescent dye and the quencher prevents light excitation and emission. In the presence of amplicon target, the complementary loop fragment of the probe is able to hybridize to the template sequence and stretches out the two ends, thus diminishing the quenching effect and resulting in detectable fluorescence (FRET does not occur).
Because the hybrid hairpin configuration is very thermostable, molecular beacons have a high specificity to hybridize to a target, which are used to distinguish single nucleotide differences. Therefore, molecular beacons are suitable for mutation analysis and single nucleotide polymorphism detection when specific mutations are known (McKilli, 2000; Szuhai, 2001; Abravaya, 2003; Bustamante, 2004; Petersen, 2004; Vet, 2005).
All real-time PCR chemistries allow detection of multiple DNA species (multiplexing) by designing each probe/beacon with a spectrally unique fluor/quench pair. All of the above can be used in conjunction to melting curve analysis or when SYBR Green is used only. By multiplexing, the target(s) and endogenous control can be amplified in a single tube. (Bernard, 1998; Lee, 1999; Vet, 1999; Elnifro, 2000; Read, 2001; Grace, 2003; Rickert, 2004)
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