Under this grant we propose to continue the development and application of a new class of fluorescence quenched probes with optimized features for genetic analysis and quantitation. The enhanced performance of our novel probes is by virtue of a set of high performance dark quenchers we refer to as """"""""Black Hole Quenchers,"""""""" or BHQs. The BHQ quenchers have been designed to optimize quenching by two independent modes, 1) formation of a ground state complex between the fluorophore and quencher leading to quenching by non-FRET mechanism, and 2) maximize spectral overlap between the fluorophore emission and quencher absorption to maximize FRET quenching, We have prepared a set of three quenchers to provide effective quenching from 480 nm to 720 nm. BHQ probes will have several desirable features which differentiate them from currently available probes. First, BHQ probes have extraordinarily low spurious noise (ie no non-specific reporter signal) and thus have a very high signal to noise ratio (S/N). This property will result in enhanced sensitivity and lower detection thresholds when compared with currently available probes, thus fewer copies of gene targets will be detectable. Secondly, BHQ probes will be more readily purified than conventional FRET probes and thus will be more cost effective to manufacture. Since BHQs are """"""""dark"""""""" quenchers they expand the available bandwidth thus allowing the multiplexing of several different probes at once. BHQ quenchers will improve the performance of virtually all FRET probe mediated assays, including Taqman, Molecular Beacons, Sunrise Scorpions, and Invader.
Self-quenching fluorogenic oligonucleotide probes have become important tools for the detection, quantitation, and discrimination of genetic matter. However current probe designs are difficult and expensive to prepare and often have extraneous noise which limits their usefulness. The new class of probes proposed here will be more economically manufactured and will have very low noise. It is estimated that probe cost will diminish from the current average of $400 per probe to less than $100 per probe, thus opening quenched probes to entirely new applications and markets.
