Wavelength-shifting probes and primers and their use in assays and kits

A fluorescently labeled hairpin-forming oligonucleotide containing:

• a) a fluorescent emitter moiety having an excitation spectrum and an emission spectrum including a maximum emission wavelength;
• b) a fluorescent harvester moiety having an excitation spectrum including a maximum excitation wavelength, having an emission spectrum that overlaps the excitation spectrum of the emitter moiety and including a maximum emission wavelength, the emission of the harvester moiety at its maximum emission wavelength having a first magnitude when said harvester moiety is unquenched and stimulated at its maximum excitation wavelength;
• and
• c) a quencher moiety capable of quenching the fluorescence of at least one of the emitter moiety and the harvester moiety,
• said oligonucleotide having a closed conformation including a single-stranded loop and a stem duplex, wherein said quencher moiety is in a quenching relationship to at least one of said harvester and emitter moieties and wherein, when excited at the maximum excitation wavelength of the harvester moiety, emission at the maximum emission wavelength of the harvester moiety is substantially suppressed relative to said first magnitude and emission at the maximum emission wavelength of the emitter moiety has a second magnitude, and
• said oligonucleotide having an open conformation not including said stem duplex in which said quencher moiety is not in a quenching relationship with said harvester or said emitter moiety wherein, when excited at the maximum excitation wavelength of the harvester moiety, emission at the maximum emission wavelength of the harvester moiety is substantially suppressed relative to said first magnitude, energy is transferred from the harvester moiety to the emitter moiety, and emission at the maximum emission wavelength of the emitter moiety is detectably greater than said second magnitude.