Dark quenchers for donor-acceptor energy transfer
AssigneeBiosearch Technologies, Inc.
InventorsRonald M. Cook, Matt Lyttle, Daren Dick
Life of this patent
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Abstract
The present invention provides a family of dark quenchers, termed Black Hole Quenchers (“BHQs”), that are efficient quenchers of excited state energy but which are themselves substantially non-fluorescent. Also provided are methods of using the BHQs, probes incorporating the BHQs and methods of using the probes.
Claims24
A method for detecting a nucleic acid target sequence, said method comprising:
- (a) contacting said target sequence with a detector nucleic acid;
- comprising a single-stranded target binding 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) hybridizing said target binding sequence to said target sequence, thereby altering said conformation of said detector nucleic acid, causing a change in a fluorescence parameter;
- and
- (c) detecting said change in said fluorescence parameter, thereby detecting said nucleic acid target sequence.
A method for detecting amplification of a target sequence comprising, in an amplification reaction:
- (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.
A method of ascertaining whether a first nucleic acid and a second nucleic acid hybridize, said first nucleic acid comprising 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, covalently linked to said nucleic acid and a second nucleic acid, said method comprising:
- (a) contacting said first nucleic acid with said second nucleic acid;
- (b) detecting an alteration in a fluorescent property of a member selected from said first nucleic acid, said second nucleic acid and a combination thereof, thereby ascertaining whether said hybridization occurs.
Dependencies are inferred from the claim text.