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Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations

med by short complementary sequences located at the opposite ends of the molecule. The molecule is labeled with a fluorophore at one end and a quencher at the other end. When the unhybridized probe is in solution, it adopts a hairpin structure that brings the fluorophore and quencher sufficiently close to each other to allow efficient quenching of the fluorophore. When the molecular beacon is bound to its complementary target, the fluorophore and quencher are far enough apart so that the fluorophore cannot be quenched, and the molecular beacon fluoresces (Figure 1). The hairpin shape of the probe causes mismatched probe/target hybrids to easily dissociate at a significantly lower temperature than perfectly matched hybrids.2

Fig.1

The thermal instability of mismatched hybrids increases the specificity of molecular beacons, enabling them to distinguish targets that differ by only a single nucleotide. When conjugated to different fluorophores, two allele-specific molecular beacons can simultaneously discriminate the three possible genotype representations (two homozygotes and a heterozygote) of two allelic variants in a single reaction tube (Figure 1).

Convenient Single-Tube Format

Each assay kit includes two allele-specific molecular beacons with an exact sequence match to each of the two target sequence variants, genotype DNA controls (wild type/mutant homozygotes and heterozygote), target-specific PCR primers, and an optimized PCR buffer. The wild-type allele-specific molecular beacon is labeled with the fluorophore tetrachloro 6-carboxyfluorescein (TET), and the mutant allele-specific molecular beacon is labeled with the fluorophore 6-carboxyfluorescein (FAM). DABCYL is used
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