Hybridization induced dequenching of fluorescein-labeled oligonucleotides - A novel strategy for PCR detection and genotyping

Fluorescence-based detection methods are being increasingly utilized in molecular analyses. Sequence-specific fluorescently-labeled probes are favored because they provide specific product identification. The most established fluorescence-based detection systems employ a resonance energy transfer mechanism effected through the interaction of two or more fluorophores or functional groups conjugated to oligonucleotide probes. The design, synthesis and purification of such multiple fluorophore-labeled probes can be technically challenging and expensive. By comparison, single fluorophore-labeled probes are easier to design and synthesize, and are straightforward to implement in molecular assays. We describe herein a novel fluorescent strategy for specific nucleic acid detection and genotyping. The format utilizes an internally quenched fluorescein-oligonucleotide conjugate that is subsequently dequenched following hybridization to the target with an attendant increase in fluorescence. Reversibility of the process with strand dissociation permits Tm-based assessment of bp complementarity and mismatches. Using this approach, we demonstrated specific detection, and discrimination of base substitutions of a variety of synthetic nucleic acid targets including Factor V Leiden and methylenetetrahydrofolate reductase. We further demonstrated compatibility of the novel chemistry with polymerase chain reaction by amplification and genotyping of the above listed loci and the human hemoglobin 0 chain locus. In total, we analyzed 172 clinical samples, comprising wild-type, heterozygous and homozygous mutants of all three loci, with 100% accuracy as confirmed by DNA sequencing, established dual hybridization probe or high performance liquid chromatography-based methods. Our results indicate that the dequenching-based single fluorophore format is a feasible strategy for the specific detection of nucleic acids in solution, and that assays using this strategy can provide accurate genotyping results.