Molecular beacons
Molecular beacons were developed as an extension of the concept of fluorescently labeled oligonucleotides. The molecular beacon is a folded probe that gives no fluorescent signal in the folded position due to quenching of the label. Upon hybridization of the molecular beacon to the target sequence (amplicon RNA), the probe unfolds and the fluorescent label emits light. When the molecular beacon is away from its target sequence, the stem structure holds the fluorphore and the quenching groups together but the stem structure is pulled apart upon interaction with target DNA. During the PCR, molecular beacons bind to the synthesized DNA and the resulting level of fluorescence can be directly correlated with the target DNA. This is an improvement on measuring the amount of PCR products by treating the sample with intercalating fluorescent dyes which lack specificity. One of the first techniques to use molecular beacons was realtime PCR. Several innovations have been made in molecular beacons. Molecular beacons may be coupled to NASBA (nucleic acid sequence-based amplification).
Molecular beacons are able to discriminate alleles in real-time PCR assays of genomic DNA. This approach can be used to analyze any DNA sequence of moderate length with single base pair accuracy. Molecular beacons present a solution for the high-throughput screening of SNPs in homogenous assays using the PCR. The ability of molecular beacons to discriminate between sequences makes them an ideal tool for genetic screening and diagnostics. Molecular beacons are also moving into the biochip world.
Single-base mismatches can be detected using DNA microarrays in a format that does not require labeling of the sample (target) DNA. The method is based on disrupting fluorescence energy transfer (FRET) between a fluorophore attached to an immobilized DNA strand (probe) and a quencher-containing sequence that is complementary. Using this method with an oligonucleotide model system, single-base mismatches can successfully discriminate at levels greater than that observed using surface-immobilized molecular beacons. A pair of molecular beacons, one with a donor and the other with an acceptor fluorophore that hybridize to adjacent regions on the same mRNA target, result in FRET. Such a dual FRET molecular beacons approach provides a novel technique for sensitive RNA detection and quantification in living cells.
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