Detection of nucleic acids by fluorescence quenching

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

• a) hybridizing to the target sequence a detector oligonucleotide comprising a single-stranded target binding sequence and an intramolecularly base-paired secondary structure 5' to the target binding sequence, wherein at least a portion of the target binding sequence forms a single-stranded tail which binds to the target sequence, the secondary structure having linked thereto a donor fluorophore and an acceptor dye such that fluorescence of the donor fluorophore is quenched;
• b) extending the hybridized detector oligonucleotide on the target sequence with a polymerase to produce a detector oligonucleotide extension product and separating the detector oligonucleotide extension product from the target sequence;
• c) hybridizing a primer to the detector oligonucleotide extension product and extending the primer with the polymerase, thereby linearizing or unfolding the secondary structure and producing a change in a fluorescence parameter, and;
• d) detecting the change in the fluorescence parameter as an indication of amplification of the target sequence.

A method for detecting a nucleic acid target sequence comprising:

• a) hybridizing to the target sequence a detector oligonucleotide comprising a single-stranded target binding sequence and an intramolecularly base-paired secondary structure 5' to the target binding sequence, wherein at least a portion of the target binding sequence forms a single-stranded tail which binds to the target sequence and the intramolecularly base-paired secondary structure comprises a partially or entirely single-stranded restriction endonuclease recognition site, the secondary structure having linked thereto a donor fluorophore and an acceptor dye such that fluorescence of the donor fluorophore is quenched;
• b) in a primer extension reaction, synthesizing a complementary strand using the base-paired secondary structure as a template, thereby linearizing or unfolding the base-paired secondary structure, rendering the restriction endonuclease recognition site partially or entirely double-stranded and producing a change in a fluorescence parameter;
• c) optionally, cleaving or nicking the partially or entirely double-stranded restriction endonuclease recognition site, and;
• d) detecting the change in the fluorescence parameter as an indication of the presence of the target sequence.

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

• a) hybridizing to the target sequence a detector oligonucleotide comprising a single-stranded target binding sequence and an intramolecularly base-paired secondary structure 5' to the target binding sequence, wherein at least a portion of the target binding sequence forms a single-stranded tail which binds to the target sequence and the intramolecularly base-paired secondary structure comprises a partially or entirely single-stranded restriction endonuclease recognition site, the secondary structure having linked thereto a donor fluorophore and an acceptor dye such that fluorescence of the donor fluorophore is quenched;
• b) extending the hybridized detector oligonucleotide on the target sequence with a polymerase to produce a detector oligonucleotide extension product and separating the detector oligonucleotide extension product from the target sequence;
• c) hybridizing a primer to the detector oligonucleotide extension product and extending the primer with a polymerase, thereby linearizing or unfolding the secondary structure, rendering the restriction endonuclease recognition site partially or entirely double-stranded and producing a change in a fluorescence parameter;
• d) optionally, cleaving or nicking the partially or entirely double-stranded restriction endonuclease recognition site, and;
• e) detecting the change in the fluorescence parameter as an indication of the presence of the target sequence.