US5723591A
Expired / Lapsed
US
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Self-quenching fluorescence probe
Assignee: Applied Biosystems LLC
•
Filing: 1995-11-15
•
Granted: 1998-03-03
•
Expires: 2015-11-15
Abstract
Abstract An oligonucleotide probe is provided which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibit different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.
Inventors
- 👤 Kenneth J. Livak
- 👤 Susan J.A. Flood
- 👤 Jeffrey Marmaro
- 👤 Khairuzzaman Bashar Mullah
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 19:27
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C12Q1/6834; C12Q1/6837
Claims Analysis
Total Claims: 47
Claim 1
Independent Claim
136 words
An oligonucleotide probe comprising: an oligonucleotide sequence which does not hybridize with itself to form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence; said oligonucleotide sequence existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide sequence existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is hybridized to said target polynucleotide is at least a factor of 6 greater than the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 2
Dependent Claim
136 words
An oligonucleotide probe comprising: an oligonucleotide sequence which does not hybridize with itself to form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence; said oligonucleotide sequence existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide sequence existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is hybridized to said target polynucleotide is at least a factor of 6 greater than the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 3
Dependent Claim
22 words
The oligonucleotide probe according to claim 1 wherein said reporter molecule is separated from said quencher molecule by at least 15 nucleotides.
Claim 4
Dependent Claim
23 words
The oligonucleotide probe according to claim 2 wherein said reporter molecule is separated from said quencher molecule by between 15 and 60 nucleotides.
Claim 5
Dependent Claim
22 words
The oligonucleotide probe according to claim 1 wherein said reporter molecule is separated from said quencher molecule by at least 18 nucleotides.
Claim 6
Dependent Claim
23 words
The oligonucleotide probe according to claim 4 wherein said reporter molecule is separated from said quencher molecule by between 18 and 30 nucleotides.
Claim 7
Dependent Claim
22 words
The oligonucleotide probe according to claim 1 wherein said reporter molecule is attached to a 3' terminal nucleotide of said oligonucleotide sequence.
Claim 8
Dependent Claim
22 words
The oligonucleotide probe according to claim 6 wherein said quencher molecule is attached to a 5' terminal nucleotide of said oligonucleotide sequence.
Claim 9
Dependent Claim
22 words
The oligonucleotide probe according to claim 1 wherein said reporter molecule is attached to a 5' terminal nucleotide of said oligonucleotide sequence.
Claim 10
Dependent Claim
22 words
The oligonucleotide probe according to claim 8 wherein said quencher molecule is attached to a 3' terminal nucleotide of said oligonucleotide sequence.
Claim 11
Dependent Claim
22 words
The oligonucleotide probe according to claim 1 wherein said quencher molecule is attached to a 3' terminal nucleotide of said oligonucleotide sequence.
Claim 12
Dependent Claim
22 words
The oligonucleotide probe according to claim 1 wherein said quencher molecule is attached to a 5' terminal nucleotide of said oligonucleotide sequence.
Claim 13
Dependent Claim
23 words
The oligonucleotide probe according to claim 1 wherein said reporter molecule is a fluorescein dye and said quencher molecule is a rhodamine dye.
Claim 14
Dependent Claim
39 words
The oligonucleotide probe according to claim 1 wherein said quencher is fluorescent and the fluorescence intensity of said reporter molecule is greater than the fluorescence intensity of said quencher molecule when said sequence is hybridized to said target polynucleotide.
Claim 15
Dependent Claim
40 words
The oligonucleotide probe according to claim 13 wherein the fluorescence intensity of said reporter molecule is at least a factor of 3.5 greater than the fluorescence intensity of said quencher molecule when said sequence is hybridized to said target polynucleotide.
Claim 16
Dependent Claim
148 words
An oligonucleotide probe comprising: an oligonucleotide sequence which does not hybridize with itself to form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a fluorescent quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence; said oligonucleotide sequence existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide sequence existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said probe is hybridized to said target polynucleotide is at least 6 times greater than the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 17
Dependent Claim
148 words
An oligonucleotide probe comprising: an oligonucleotide sequence which does not hybridize with itself to form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a fluorescent quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence; said oligonucleotide sequence existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide sequence existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said probe is hybridized to said target polynucleotide is at least 6 times greater than the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 18
Dependent Claim
138 words
An oligonucleotide probe comprising: a solid support; an oligonucleotide sequence attached to said solid support which does not form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence; said oligonucleotide sequence existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide sequence existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is hybridized to said target polynucleotide is at least 6 times greater than the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 19
Dependent Claim
138 words
An oligonucleotide probe comprising: a solid support; an oligonucleotide sequence attached to said solid support which does not form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence; said oligonucleotide sequence existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide sequence existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is hybridized to said target polynucleotide is at least 6 times greater than the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 20
Dependent Claim
19 words
The probe according to claim 16 wherein said oligonucleotide sequence is attached to said solid support by a linker.
Claim 21
Dependent Claim
22 words
The probe according to claim 17 wherein said linker separates said oligonucleotide sequence from said solid support by at least 30 atoms.
Claim 22
Dependent Claim
22 words
The probe according to claim 18 wherein said linker separates said oligonucleotide sequence from said solid support by at least 50 atoms.
Claim 23
Dependent Claim
14 words
The probe according to claim 17 wherein the linker is a functionalized polyethylene glycol.
Claim 24
Dependent Claim
12 words
The probe according to claim 20 wherein the linker is a polynucleotide.
Claim 25
Dependent Claim
24 words
The probe according to claim 17 wherein said linker includes a detachable linkage enabling said oligonucleotide sequence to be released from said solid support.
Claim 26
Dependent Claim
38 words
The probe according to claim 16 wherein said quencher is fluorescent and the fluorescence intensity of said reporter molecule is greater than the fluorescence intensity of said quencher molecule when said sequence is hybridized to said target polynucleotide.
Claim 27
Dependent Claim
39 words
The probe according to claim 23 wherein the fluorescence intensity of said reporter molecule is at least a factor of 3.5 greater than the fluorescence intensity of said quencher molecule when said sequence is hybridized to said target polynucleotide.
Claim 28
Dependent Claim
152 words
An oligonucleotide probe comprising: a solid support; an oligonucleotide sequence attached to said solid support which does not form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a fluorescent quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence; said oligonucleotide probe existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said probe is hybridized to said target polynucleotide is at least 6 times greater than the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 29
Dependent Claim
152 words
An oligonucleotide probe comprising: a solid support; an oligonucleotide sequence attached to said solid support which does not form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a fluorescent quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence; said oligonucleotide probe existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said probe is hybridized to said target polynucleotide is at least 6 times greater than the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 30
Dependent Claim
146 words
An oligonucleotide probe comprising: an oligonucleotide sequence which does not hybridize with itself to form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence such that said quencher molecule is separated from said reporter molecule by at least 15 nucleotides; said oligonucleotide sequence existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide sequence existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is hybridized to said target polynucleotide is greater than the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 31
Dependent Claim
146 words
An oligonucleotide probe comprising: an oligonucleotide sequence which does not hybridize with itself to form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence such that said quencher molecule is separated from said reporter molecule by at least 15 nucleotides; said oligonucleotide sequence existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide sequence existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is hybridized to said target polynucleotide is greater than the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 32
Dependent Claim
22 words
The oligonucleotide probe according to claim 26 wherein said reporter molecule is separated from said quencher molecule by at least 18 nucleotides.
Claim 33
Dependent Claim
23 words
The oligonucleotide probe according to claim 26 wherein said reporter molecule is separated from said quencher molecule by between 15 and 60 nucleotides.
Claim 34
Dependent Claim
23 words
The oligonucleotide probe according to claim 26 wherein said reporter molecule is separated from said quencher molecule by between 18 and 60 nucleotides.
Claim 35
Dependent Claim
23 words
The oligonucleotide probe according to claim 26 wherein said reporter molecule is separated from said quencher molecule by between 18 and 30 nucleotides.
Claim 36
Dependent Claim
153 words
A probe comprising: a solid support; an oligonucleotide sequence attached to said solid support which does not hybridize with itself to form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence such that said quencher molecule is separated from said reporter molecule by at least 15 nucleotides; said oligonucleotide sequence existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide sequence existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is hybridized to said target polynucleotide is greater than the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 37
Dependent Claim
153 words
A probe comprising: a solid support; an oligonucleotide sequence attached to said solid support which does not hybridize with itself to form a hairpin structure; a fluorescent reporter molecule attached to said oligonucleotide sequence; and a quencher molecule capable of quenching the fluorescence of said reporter molecule attached to said oligonucleotide sequence such that said quencher molecule is separated from said reporter molecule by at least 15 nucleotides; said oligonucleotide sequence existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide sequence existing in at least one conformation when hybridized to a target polynucleotide where the fluorescence of said reporter molecule is unquenched, the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is hybridized to said target polynucleotide is greater than the fluorescence intensity of said reporter molecule when said oligonucleotide sequence is not hybridized to said target polynucleotide.
Claim 38
Dependent Claim
21 words
The probe according to claim 31 wherein said reporter molecule is separated from said quencher molecule by at least 18 nucleotides.
Claim 39
Dependent Claim
22 words
The probe according to claim 31 wherein said reporter molecule is separated from said quencher molecule by between 15 and 60 nucleotides.
Claim 40
Dependent Claim
22 words
The probe according to claim 31 wherein said reporter molecule is separated from said quencher molecule by between 18 and 60 nucleotides.
Claim 41
Dependent Claim
22 words
The probe according to claim 31 wherein said reporter molecule is separated from said quencher molecule by between 18 and 30 nucleotides.
Claim 42
Dependent Claim
18 words
The probe according to claim 31 wherein the sequence is attached to said solid support by a linker.
Claim 43
Dependent Claim
22 words
The probe according to claim 36 wherein said linker separates said oligonucleotide sequence from said solid support by at least 30 atoms.
Claim 44
Dependent Claim
22 words
The probe according to claim 36 wherein said linker separates said oligonucleotide sequence from said solid support by at least 50 atoms.
Claim 45
Dependent Claim
14 words
The probe according to claim 36 wherein said linker is a functionalized polyethylene glycol.
Claim 46
Dependent Claim
12 words
The probe according to claim 39 wherein said linker is a polynucleotide.
Claim 47
Dependent Claim
24 words
The probe according to claim 36 wherein said linker includes a detachable linkage enabling said oligonucleotide sequence to be released from said solid support.
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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) | 2001-03-02 | 2001-09-01 | 2002-03-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-03-02 | 2005-09-01 | 2006-03-03 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-03-02 | 2009-09-01 | 2010-03-03 | $7700.00 | Window Closed / Lapsed |