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 and a single-stranded restriction endonuclease recognition sequence 5'"'"' to the target binding sequence, the restriction endonuclease recognition sequence flanked by 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 signal primer as a template, thereby rendering the restriction endonuclease recognition sequence 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.
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Citations
51 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 and a single-stranded restriction endonuclease recognition sequence 5'"'"' to the target binding sequence, the restriction endonuclease recognition sequence flanked by 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 signal primer as a template, thereby rendering the restriction endonuclease recognition sequence 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)
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13. 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 comprising a target binding sequence and a restriction endonuclease recognition sequence 5'"'"' to the target binding sequence, the restriction endonuclease recognition sequence flanked by a donor fluorophore and an acceptor dye such that fluorescence of the donor fluorophore is quenched; b) extending the hybridized first primer on the target sequence with a polymerase 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 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 (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
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34. 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 comprising a donor fluorophore and an acceptor dye, the donor fluorophore and the acceptor dye linked to the first primer such that fluorescence of the donor fluorophore is quenched; b) extending the hybridized first primer on the target sequence with a polymerase 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 double-stranded by hybridization and extension of a second primer, 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 amplification of the target sequence. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
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47. A single-stranded oligonucleotide comprising:
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(a) a target binding sequence; (b) a restriction endonuclease recognition site 5'"'"' to the target binding 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 (48, 49, 50, 51)
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Specification