Patent ID: 8440399

Claim:
A method for detecting a nucleic acid target sequence, said method comprising: amplifying said nucleic acid target sequence, wherein the amplification reaction comprises the steps of: (a) hybridizing to said target sequence a detector nucleic acid comprising a single-stranded target binding sequence and an intramolecularly associated secondary structure 5′ to said target binding sequence, wherein at least a portion of said detector sequence forms a single stranded tail which is available for hybridization to said target sequence, said detector nucleic acid having linked thereto, i) a fluorophore; and ii) a quencher of excited state energy having a structure comprising at least three residues selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl and combinations thereof, wherein at least two of said residues are covalently linked via an exocyclic diazo bond, wherein said detector nucleic acid is in a conformation allowing donor-acceptor energy transfer between said fluorophore and said quencher when said fluorophore is excited; (b) extending said hybridized detector nucleic acid on said target sequence with a polymerase to produce a detector nucleic acid extension product and separating said detector nucleic acid extension product from said target sequence; (c) hybridizing a primer to said detector nucleic acid extension product and extending the primer with said polymerase, thereby linearizing said intramolecularly associated secondary structure and producing a change in a fluorescence parameter; and (d) detecting said change in said fluorescence parameter, thereby detecting said target sequence.