Conformationally Assisted Fet Probes

Cook, Ronald

Abstract

Under this grant we propose to develop a new class of self-quenching fluorescence energy transfer probes (FET) with optimized features for genetic discrimination and quantitation. The enhanced performance of our novel probes is by virtue of appended affinity ligands which lead to favorable oligonucleotide conformations, resulting in enhanced interaction of the fluorophore/quencher pair We refer to these new probes as Conformationally Assisted Probes or CAP probes. CAP probes will have several desirable features which differentiate them from currently available probes. First, CAP 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, CAP probes will be more readily purified than conventional FET probes and thus will be more cost effective to manufacture. Further potential includes the use of CAP probes as superior agents for in vivo real time expression analysis, and as immobilized probes for detection of targets in a microarray format.

Proposed Commercial Applications

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 FET probe use to entirely new applications and markets.