Universal probes and methods for detection of nucleic acids

A method for detecting amplification of a target sequence comprising, in an amplification reaction:

• a) hybridizing to the target sequence a signal primer comprising
• i) a reporter probe which in the absence of hybridization to a complementary sequence assumes a conformational structure which brings a fluorescent donor/quencher dye pair linked thereto into sufficiently close spatial proximity to quench donor fluorescence, and,
• ii) an adapter oligonucleotide hybridized to the reporter probe such that the signal primer comprises an intermolecularly base-paired portion and a single-stranded target binding sequence,
• wherein quenching of donor fluorescence is greater in the absence of hybridization of the adapter oligonucleotide and the reporter probe than when the adapter oligonucleotide and the reporter probe are hybridized;
• b) extending the adapter oligonucleotide on the target sequence with a polymerase to produce an extension product and separating the extension product from the target sequence;
• c) hybridizing an amplification primer to the extension product and extending the amplification primer with the polymerase, whereby the reporter probe is separated from the adapter oligonucleotide and quenching of donor fluorescence is increased, and;
• d) detecting a change in a fluorescence parameter associated with increased quenching as an indication of amplification of the target sequence.

A method for detecting the presence of a nucleic acid target sequence comprising:

• a) hybridizing to the target sequence a signal primer comprising a first oligonucleotide hybridized to a second oligonucleotide such that the signal primer comprises an intermolecularly base-paired portion and a single-stranded target binding sequence, wherein the second oligonucleotide;
• b) in a primer extension reaction, synthesizing a strand complementary to the first oligonucleotide, whereby the second oligonucleotide is separated from the first oligonucleotide;
• c) hybridizing the separated second oligonucleotide to a reporter probe which in the absence of hybridization to a complementary sequence assumes a conformational structure which brings a fluorescent donor/quencher dye pair linked thereto into sufficiently close spatial proximity to quench donor fluorescence, and wherein quenching of donor fluorescence when the reporter probe and the second oligonucleotide are hybridized is less than quenching of donor fluorescence in the absence of hybridization of the reporter probe and the second oligonucleotide, and;
• d) detecting a change in a fluorescence parameter associated with decreased quenching as an indication of the presence of the target sequence.