Detection of nucleic acids by fluorescence quenching
First Claim
1. A method for detecting the presence of 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, 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 and producing a change in a fluorescence parameter, and;
c) detecting the change in the fluorescence parameter as an indication of the presence of the target sequence.
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Accused Products
Abstract
A detector oligonucleotide having a sequence which forms an intramolecularly base-paired secondary structure is described for use in detecting nucleic acid target sequences and target sequence amplification. The detector oligonucleotide is further modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned on the detector oligonucleotide such that they are in close spatial proximity in the base-paired, folded secondary structure, thereby causing quenching of donor fluorescence. The detector oligonucleotide may optionally further comprise a restriction endonuclease recognition site (RERS) which remains partially or entirely single-stranded in the base-paired secondary structure. The RERS is flanked by the two dyes. In the presence of target, the base-paired secondary structure is unfolded or linearized, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence of the donor and/or the acceptor. If an RERS is present, it is rendered double-stranded in the presence of target, allowing cleavage or nicking by a restriction endonuclease and separation of the two dyes onto separate nucleic acid fragments. This may further contribute to the magnitude of the change in fluorescence.
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Citations
74 Claims
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1. A method for detecting the presence of a nucleic acid target sequence comprising:
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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) 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 and producing a change in a fluorescence parameter, and; c) detecting the change in the fluorescence parameter as an indication of the presence of the target sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 48, 49, 50, 51, 52, 53, 54, 55)
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16. A method for detecting amplification of a target sequence comprising, in an amplification reaction:
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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. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 56, 57, 58, 59, 60, 61, 62)
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30. A method for detecting a target sequence comprising:
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a) providing a detector oligonucleotide comprising a single-stranded target binding sequence and an intramolecularly base-paired secondary structure adjacent to the target binding sequence, wherein at least a portion of the target binding sequence forms a single-stranded 5'"'"' or 3'"'"' 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) hybridizing the detector oligonucleotide to the target sequence, thereby reducing donor fluorophore quenching and producing a change in a fluorescence parameter, and; c) detecting the change in the fluorescence parameter as an indication of the presence of the target sequence. - View Dependent Claims (31, 32, 33, 34, 35, 36, 37, 38, 63, 64, 65, 66, 67, 68)
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- 39. An oligonucleotide comprising a single-stranded target binding sequence and an intramolecularly base-paired secondary structure adjacent to the target binding sequence, wherein at least a portion of the target binding sequence forms a single-stranded 5'"'"' or 3'"'"' tail which binds to a target sequence, the secondary structure having linked thereto a first dye and a second dye such that fluorescence of the first or the second dye is quenched in the intramolecularly base-paired secondary structure and a change in a fluorescence parameter is detectable upon linearization or unfolding of the secondary structure.
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69. A method for detecting a nucleic acid target sequence comprising:
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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. - View Dependent Claims (70, 71)
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72. A method for detecting amplification of a target sequence comprising, in an amplification reaction:
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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. - View Dependent Claims (73, 74)
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Specification