Methods and compositions for detection of nucleic acids based on stabilized oligonucleotide probe complexes

Abstract

Provided are nucleic acid detection methods wherein targeted primer extension or products or amplification products incorporate a probe-anchoring modification introduced using a primer incorporating a probe-anchoring primer modification, wherein oligonucleotide detection probes incorporate a probe-anchoring probe modification, the primers and probes designed to place the binding site of the oligonucleotide probe proximate to the probe-anchoring primer modification in the detected target sequence. The probe-anchoring modifications of the probe and the primer-extension product form a stabilized complex comprising a detectible duplex of the probe with the detected target sequence. In certain aspects, the probe-anchoring modified primer also incorporates a probe-directing sequence. The methods allow use of exceptionally short oligonucleotide probes (e.g., 10-mer and shorter) enabling establishment of a complete probe inventory or universal library containing a probe complementary to any target nucleic acid sequence. Real-time and post-amplification detection methods are provided, along with detection kits.