US5846726A
Expired / Lapsed
US
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Detection of nucleic acids by fluorescence quenching
Assignee: Becton Dickinson and Co
•
Filing: 1997-05-13
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Granted: 1998-12-08
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Expires: 2017-05-13
Abstract
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.
Inventors
- 👤 James G. Nadeau
- 👤 J. Bruce Pitner
- 👤 James L. Schram
- 👤 C. Preston Linn
- 👤 Glenn P. Vonk
- 👤 G. Terrance Walker
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 04:09
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C12Q1/6825
Claims Analysis
Total Claims: 55
Claim 1
Independent Claim
131 words
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.
Claim 2
Independent Claim
131 words
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.
Claim 3
Dependent Claim
16 words
The method of claim 1 wherein the complementary strand is synthesized in a target amplification reaction.
Claim 4
Dependent Claim
24 words
The method of claim 1 wherein the complementary strand is synthesized by extension of the target sequence using the signal primer as a template.
Claim 5
Dependent Claim
23 words
The method of claim 1 wherein a change in fluorescence intensity is detected as an indication of the presence of the target sequence.
Claim 6
Dependent Claim
33 words
The method of claim 4 wherein an increase in donor fluorophore fluorescence intensity or a decrease in acceptor dye fluorescence intensity is detected as an indication of the presence of the target sequence.
Claim 7
Dependent Claim
60 words
The method of claim 5 wherein the change in fluorescence intensity is detected as i) an increase in a ratio of donor fluorophore fluorescence after cleaving or nicking to donor fluorophore fluorescence in the signal primer, or ii) as a decrease in a ratio of acceptor dye fluorescence after cleaving or nicking to acceptor dye fluorescence in the signal primer.
Claim 8
Dependent Claim
31 words
The method of claim 1 wherein a change in fluorescence lifetime of the donor fluorophore or the acceptor dye is detected as an indication of the presence of the target sequence.
Claim 9
Dependent Claim
16 words
The method of claim 1 wherein the change in the fluorescence parameter is detected in real-time.
Claim 10
Dependent Claim
17 words
The method of claim 1 wherein the change in the fluorescence parameter is detected at an endpoint.
Claim 11
Dependent Claim
23 words
The method of claim 1 wherein the donor fluorophore and the acceptor dye are separated by about 8-20 nucleotides in the signal primer.
Claim 12
Dependent Claim
23 words
The method of claim 10 wherein the donor fluorophore and the acceptor dye are separated by about 10-16 nucleotides in the signal primer.
Claim 13
Dependent Claim
18 words
The method of claim 1 wherein the donor fluorophore is fluorescein and the acceptor dye is Rhodamine X.
Claim 14
Independent Claim
162 words
A method for detecting amplification of a target sequence comprising, in an amplification reaction: 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.
Claim 15
Independent Claim
162 words
A method for detecting amplification of a target sequence comprising, in an amplification reaction: 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.
Claim 16
Dependent Claim
15 words
The method of claim 13 wherein the target sequence is amplified by Strand Displacement Amplification.
Claim 17
Dependent Claim
22 words
The method of claim 14 further comprising nicking of the double-stranded restriction endonuclease recognition sequence in an amplification primer during target amplification.
Claim 18
Dependent Claim
13 words
The method of claim 15 wherein the restriction endonuclease is BsoBI or Aval.
Claim 19
Dependent Claim
27 words
The method of claim 14 further comprising a second restriction endonuclease which nicks a second double-stranded restriction endonuclease recognition sequence in an amplification primer during target amplification.
Claim 20
Dependent Claim
20 words
The method of claim 13 wherein the target sequence is amplified by the Polymerase Chain Reaction, 3SR, TMA or NASBA.
Claim 21
Dependent Claim
13 words
The method of claim 13 wherein the first primer is a signal primer.
Claim 22
Dependent Claim
13 words
The method of claim 19 wherein the second primer is an amplification primer.
Claim 23
Dependent Claim
14 words
The method of claim 13 wherein the first and second primers are amplification primers.
Claim 24
Dependent Claim
16 words
The method of claim 13 wherein the change in the fluorescence parameter is detected in real-time.
Claim 25
Dependent Claim
22 words
The method of claim 13 wherein the change in the fluorescence parameter is detected at a selected end-point in the amplification reaction.
Claim 26
Dependent Claim
23 words
The method of claim 13 wherein the donor fluorophore and the acceptor dye are separated by about 8-20 nucleotides in the first primer.
Claim 27
Dependent Claim
23 words
The method of claim 24 wherein the donor fluorophore and the acceptor dye are separated by about 10-16 nucleotides in the first primer.
Claim 28
Dependent Claim
18 words
The method of claim 13 wherein the donor fluorophore is fluorescein and the acceptor dye is Rhodamine X.
Claim 29
Dependent Claim
22 words
The method of claim 26 wherein the donor fluorophore and the acceptor dye are separated by 11 nucleotides in the first primer.
Claim 30
Dependent Claim
17 words
The method of claim 13 wherein the donor fluorophore is fluorescein and the acceptor dye is DABCYL.
Claim 31
Dependent Claim
22 words
The method of claim 28 wherein the donor fluorophore and the acceptor dye are separated by 11 nucleotides in the first primer.
Claim 32
Dependent Claim
18 words
The method of claim 13 wherein the donor fluorophore is Rhodamine X and the acceptor dye is Cy5.
Claim 33
Dependent Claim
22 words
The method of claim 30 wherein the donor fluorophore and the acceptor dye are separated by 11 nucleotides in the first primer.
Claim 34
Dependent Claim
27 words
The method of claim 13 wherein a change in donor fluorophore or acceptor dye fluorescence intensity is detected as an indication of amplification of the target sequence.
Claim 35
Dependent Claim
61 words
The method of claim 32 wherein the change in the fluorescence intensity is detected i) as an increase in a ratio of donor fluorophore fluorescence after cleaving or nicking to donor fluorophore fluorescence in the first primer, or ii) as a decrease in a ratio of acceptor dye fluorescence after cleaving or nicking to acceptor dye fluorescence in the first primer.
Claim 36
Independent Claim
131 words
A method for detecting amplification of a target sequence comprising, in an amplification reaction: 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.
Claim 37
Independent Claim
131 words
A method for detecting amplification of a target sequence comprising, in an amplification reaction: 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.
Claim 38
Dependent Claim
18 words
The method of claim 34 wherein the donor fluorophore and the acceptor dye are an homologous dye pair.
Claim 39
Dependent Claim
27 words
The method of claim 35 wherein a change in donor fluorophore or acceptor dye fluorescence intensity is detected as an indication of amplification of the target sequence.
Claim 40
Dependent Claim
66 words
The method of claim 36 wherein the change in fluorescence intensity is detected i) as an increase in a ratio of donor fluorophore fluorescence in the double-stranded first primer extension product to donor fluorophore fluorescence in the first primer, or ii) as a decrease in a ratio of acceptor dye fluorescence in the double-stranded first primer extension product to acceptor dye fluorescence in the first primer.
Claim 41
Dependent Claim
25 words
The method of claim 35 wherein the donor fluorophore and the acceptor dye are selected from the group consisting of Cy5, Rhodamine X and fluorescein.
Claim 42
Dependent Claim
16 words
The method of claim 38 wherein the donor fluorophore and the acceptor dye are Rhodamine X.
Claim 43
Dependent Claim
18 words
The method of claim 34 wherein the donor fluorophore and the acceptor dye are a heterologous dye pair.
Claim 44
Dependent Claim
27 words
The method of claim 40 wherein a change in donor fluorophore or acceptor dye fluorescence intensity is detected as an indication of amplification of the target sequence.
Claim 45
Dependent Claim
66 words
The method of claim 41 wherein the change in fluorescence intensity is detected i) as an increase in a ratio of donor fluorophore fluorescence in the double-stranded first primer extension product to donor fluorophore fluorescence in the first primer, or ii) as a decrease in a ratio of acceptor dye fluorescence in the double-stranded first primer extension product to acceptor dye fluorescence in the first primer.
Claim 46
Dependent Claim
20 words
The method of claim 40 wherein the donor fluorophore is fluorescein and the acceptor dye is Rhodamine X or DABCYL.
Claim 47
Dependent Claim
13 words
The method of claim 34 wherein the first primer is a signal primer.
Claim 48
Dependent Claim
13 words
The method of claim 44 wherein the second primer is an amplification primer.
Claim 49
Dependent Claim
14 words
The method of claim 34 wherein the first and second primers are amplification primers.
Claim 50
Dependent Claim
54 words
A single-stranded oligonucleotide comprising: (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.
Claim 51
Dependent Claim
54 words
A single-stranded oligonucleotide comprising: (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.
Claim 52
Dependent Claim
19 words
The oligonucleotide of claim 47 wherein the first and second dyes are about 8-20 nucleotides apart in the oligonucleotide.
Claim 53
Dependent Claim
19 words
The oligonucleotide of claim 48 wherein the first and second dyes are about 10-16 nucleotides apart in the oligonucleotide.
Claim 54
Dependent Claim
18 words
The oligonucleotide of claim 49 wherein the first and second dyes are 11 nucleotides apart in the oligonucleotide.
Claim 55
Dependent Claim
20 words
The oligonucleotide of claim 47 wherein the first dye is fluorescein and the second dye is Rhodamine X or DABCYL.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-12-07 | 2002-06-08 | 2002-12-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-12-07 | 2006-06-08 | 2006-12-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-12-07 | 2010-06-08 | 2010-12-08 | $7700.00 | Window Closed / Lapsed |