US6030787A
Expired / Lapsed
US
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Hybridization assay using self-quenching fluorescence probe
Assignee: Applied Biosystems LLC
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Filing: 1998-12-07
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Granted: 2000-02-29
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Expires: 2018-12-07
Abstract
Abstract A hybridization assay is provided which uses an oligonucleotide probe 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 exhibits 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 Mamoro
- 👤 Khairuzzaman Bashar Mullah
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 08:41
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C12Q1/6834; C12Q1/6837
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
203 words
A method for detecting a target polynucleotide in a sample comprising: contacting said sample of nucleic acids with an oligonucleotide probe under conditions where said oligonucleotide probe selectively hybridizes to said target polynucleotide, said oligonucleotide probe including a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of said reporter molecule which are attached to said oligonucleotide probe such that said oligonucleotide probe is capable of adopting at least one single-stranded conformation when not hybridized to said target polynucleotide where said quencher molecule is fluorescent and quenches the fluorescence of said reporter molecule and is capable of adopting at least one conformation when hybridized to said target polynucleotide, where the fluorescence of said reporter molecule is unquenched such that the fluorescence intensity of said reporter molecule is greater than the fluorescence intensity of said quencher molecule when said oligonucleotide probe is hybridized to said target polynucleotide and said oligonucleotide probe is not hybridized with itself in the form of a hairpin structure; and monitoring the fluorescence of said reporter molecule under conditions where said oligonucleotide probe does not hybridize with itself to form a hairpin structure in order to detect the hybridization of said target polynucleotide to said oligonucleotide probe.
Claim 2
Independent Claim
203 words
A method for detecting a target polynucleotide in a sample comprising: contacting said sample of nucleic acids with an oligonucleotide probe under conditions where said oligonucleotide probe selectively hybridizes to said target polynucleotide, said oligonucleotide probe including a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of said reporter molecule which are attached to said oligonucleotide probe such that said oligonucleotide probe is capable of adopting at least one single-stranded conformation when not hybridized to said target polynucleotide where said quencher molecule is fluorescent and quenches the fluorescence of said reporter molecule and is capable of adopting at least one conformation when hybridized to said target polynucleotide, where the fluorescence of said reporter molecule is unquenched such that the fluorescence intensity of said reporter molecule is greater than the fluorescence intensity of said quencher molecule when said oligonucleotide probe is hybridized to said target polynucleotide and said oligonucleotide probe is not hybridized with itself in the form of a hairpin structure; and monitoring the fluorescence of said reporter molecule under conditions where said oligonucleotide probe does not hybridize with itself to form a hairpin structure in order to detect the hybridization of said target polynucleotide to said oligonucleotide probe.
Claim 3
Dependent Claim
40 words
The method according to claim 1 wherein the fluorescence intensity of said reporter molecule is at least about a factor of 3.5 greater than the fluorescence intensity of said quencher molecule when said probe is hybridized to said target polynucleotide.
Claim 4
Dependent Claim
21 words
The method according to claim 1 wherein said reporter molecule is separated from said quencher molecule by at least 15 nucleotides.
Claim 5
Dependent Claim
22 words
The method according to claim 1 wherein said reporter molecule is separated from said quencher molecule by between 15 and 60 nucleotides.
Claim 6
Dependent Claim
21 words
The method according to claim 1 wherein said reporter molecule is separated from said quencher molecule by at least 18 nucleotides.
Claim 7
Dependent Claim
22 words
The method according to claim 1 wherein said reporter molecule is separated from said quencher molecule by between 18 and 30 nucleotides.
Claim 8
Independent Claim
209 words
A method for detecting a target polynucleotide in a sample comprising: contacting said sample of nucleic acids with an oligonucleotide probe attached to a solid support under conditions favorable for hybridization of said oligonucleotide probe to said target polynucleotide, said oligonucleotide probe including a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of said reporter molecule which are attached to said oligonucleotide probe such that said oligonucleotide probe is capable of adopting at least one single-stranded conformation when not hybridized to said target polynucleotide where said quencher molecule is fluorescent and quenches the fluorescence of said reporter molecule and is capable of adopting at least one conformation when hybridized to said target polynucleotide, where the fluorescence of said reporter molecule is unquenched such that the fluorescence intensity of said reporter molecule is greater than the fluorescence intensity of said quencher molecule when said oligonucleotide probe is hybridized to said target polynucleotide and said oligonucleotide probe is not hybridized with itself in the form of a hairpin structure; and monitoring the fluorescence of said reporter molecule under conditions where said oligonucleotide probe does not hybridize with itself to form a hairpin structure in order to detect the hybridization of said target polynucleotide to said oligonucleotide probe.
Claim 9
Independent Claim
209 words
A method for detecting a target polynucleotide in a sample comprising: contacting said sample of nucleic acids with an oligonucleotide probe attached to a solid support under conditions favorable for hybridization of said oligonucleotide probe to said target polynucleotide, said oligonucleotide probe including a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of said reporter molecule which are attached to said oligonucleotide probe such that said oligonucleotide probe is capable of adopting at least one single-stranded conformation when not hybridized to said target polynucleotide where said quencher molecule is fluorescent and quenches the fluorescence of said reporter molecule and is capable of adopting at least one conformation when hybridized to said target polynucleotide, where the fluorescence of said reporter molecule is unquenched such that the fluorescence intensity of said reporter molecule is greater than the fluorescence intensity of said quencher molecule when said oligonucleotide probe is hybridized to said target polynucleotide and said oligonucleotide probe is not hybridized with itself in the form of a hairpin structure; and monitoring the fluorescence of said reporter molecule under conditions where said oligonucleotide probe does not hybridize with itself to form a hairpin structure in order to detect the hybridization of said target polynucleotide to said oligonucleotide probe.
Claim 10
Dependent Claim
40 words
The method according to claim 7 wherein the fluorescence intensity of said reporter molecule is at least about a factor of 3.5 greater than the fluorescence intensity of said quencher molecule when said probe is hybridized to said target polynucleotide.
Claim 11
Dependent Claim
21 words
The method according to claim 8 wherein said reporter molecule is separated from said quencher molecule by at least 15 nucleotides.
Claim 12
Dependent Claim
22 words
The method according to claim 8 wherein said reporter molecule is separated from said quencher molecule by between 15 and 60 nucleotides.
Claim 13
Dependent Claim
21 words
The method according to claim 8 wherein said reporter molecule is separated from said quencher molecule by at least 18 nucleotides.
Claim 14
Dependent Claim
22 words
The method according to claim 8 wherein said reporter molecule is separated from said quencher molecule by between 18 and 30 nucleotides.
References Cited (Backward Citations)
US4220450A
Cited
EP0229943B1
Cited
EP0343955A2
Cited
WO1990003446A1
Cited
US5210015A
Cited
WO1992002638A1
Cited
EP0523557A1
Cited
JPH05123195A
Cited
WO1993013224A1
Cited
US5332659A
Cited
EP0601889A2
Cited
WO1995003429A1
Cited
US5491063A
Cited
US5876930A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2003-02-28 | 2003-08-30 | 2004-02-29 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-02-28 | 2007-08-30 | 2008-02-29 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-02-28 | 2011-08-30 | 2012-02-29 | $7700.00 | Window Closed / Lapsed |