Detection of nucleic acids by fluorescence quenching
First Claim
1. A method for detecting presence of a target sequence comprising:
- a) hybridizing to the target sequence a signal primer comprising a target binding sequence, a single-stranded restiction endonuclease recognition sequence 5'"'"' to the target binding sequence and as donor fluoropore and an acceptor dye flanking the restriction endonuclease recognition sequence such that fluorescence of the donor fluorophore is quenched, wherein all or part of the restriction endonuclease recognition sequence remains single stranded upon hybridization of the signal primer to the target sequence;
b) in a primer extension reaction, synthesizing a complementary strand using the signal primer as a template, thereby rendering the restriction endonuclease recognition sequence completely double-stranded;
c) cleaving or nicking the double-stranded restriction endonuclease recognition sequence with a restriction endonuclease, thereby reducing donor fluorophore quenching and producing a change in a fluorescence parameter, and;
d) detecting the change in the fluorescence parameter as an indication of the presence of the target sequence.
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Accused Products
Abstract
Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.
97 Citations
41 Claims
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1. A method for detecting presence of a target sequence comprising:
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a) hybridizing to the target sequence a signal primer comprising a target binding sequence, a single-stranded restiction endonuclease recognition sequence 5'"'"' to the target binding sequence and as donor fluoropore and an acceptor dye flanking the restriction endonuclease recognition sequence such that fluorescence of the donor fluorophore is quenched, wherein all or part of the restriction endonuclease recognition sequence remains single stranded upon hybridization of the signal primer to the target sequence; b) in a primer extension reaction, synthesizing a complementary strand using the signal primer as a template, thereby rendering the restriction endonuclease recognition sequence completely double-stranded; c) cleaving or nicking the double-stranded restriction endonuclease recognition sequence with a restriction endonuclease, thereby reducing donor fluorophore quenching and producing a change in a fluorescence parameter, and; d) 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)
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14. 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 first primer comnprising a target binding sequence, a restriction endonuclease recognition sequence 5'"'"' to the target binding sequence and a donor fluorophore and an acceptor dye flanking the restriction endonuclease recognition sequence such that fluorescence of the donor fluorophore is quenched, wherein all or part of the restriction endonuclease recognition sequence remains single stranded upon hybridization of the signal primer to the target sequence; b) extending the hybridized first primer on the target sequence with a polyrmerase to produce a first primer extension product and separating the first primer extension product from the target sequence; c) rendering the separated first primer extension product and the restriction endonuclease recognition sequence completely double-stranded by hybridization and extension of a second primer; d) cleaving or nicking the double-stranded restriction endonuclease recognition sequence with a restriction endonuclease, thereby reducing donor fluorophore quenching and producing a change in a fluorescence parameter, and; e) detecting the change in the fluorescence parameter as an indication of amplification of the target sequence. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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36. A single-stranded oligonucleotide cormprising:
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(a) a target binding sequence; (b) a restriction endonuclease recognition site 5'"'"' to the target binding sequence such that all or part of the restriction endonuclease recognition site remains single-stranded upon hybridization of the oligonucleotide to a target sequence and; (c) a first dye and a second dye linked to the oligonucleotide at positions flanking the restriction endonuclease recognition site such that fluorescence of the first or the second dye is quenched. - View Dependent Claims (37, 38, 39, 40, 41)
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Specification