US6177249B1
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Method for nucleic acid analysis using fluorescence resonance energy transfer
Assignee: Washington University in St. Louis
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Filing: 1999-04-20
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Granted: 2001-01-23
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Expires: 2019-04-20
Abstract
Abstract A method for detecting the presence of a target nucleotide or sequence of nucleotides in a nucleic acid is disclosed. The method is comprised of forming an oligonucleotide labeled with two fluorophores on the nucleic acid target site. The doubly labeled oligonucleotide is formed by addition of a singly labeled dideoxynucleoside triphosphate to a singly labeled polynucleotide or by ligation of two singly labeled polynucleotides. Detection of fluorescence resonance energy transfer upon denaturation indicates the presence of the target. Kits are also provided. The method is particularly applicable to genotyping.
Inventors
- 👤 Pui-Yan Kwok
- 👤 Xiangning Chen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 21:20
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C12Q2600/00; C12Q2600/156
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
99 words
A kit for detecting the presence of a target site of at least one nucleotide in a nucleic acid, the kit comprising: (a) a polynucleotide covalently linked to one fluorophore and capable of binding to the target site or immediately 3′ to the target site; and (b) a dideoxynucleotide covalently linked to a second fluorophore, which is capable of binding to the nucleic acid immediately 5′ to the polynucleotide and capable of adding by 3′ extension to the polynucleotide, wherein the emission spectrum of one of the fluorophores overlaps the excitation spectrum of the other, packaged in a container.
Claim 2
Dependent Claim
99 words
A kit for detecting the presence of a target site of at least one nucleotide in a nucleic acid, the kit comprising: (a) a polynucleotide covalently linked to one fluorophore and capable of binding to the target site or immediately 3′ to the target site; and (b) a dideoxynucleotide covalently linked to a second fluorophore, which is capable of binding to the nucleic acid immediately 5′ to the polynucleotide and capable of adding by 3′ extension to the polynucleotide, wherein the emission spectrum of one of the fluorophores overlaps the excitation spectrum of the other, packaged in a container.
Claim 3
Dependent Claim
50 words
The kit according to claim 1 wherein one fluorophore is selected from the group consisting of fluorescein, cascade blue, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid and 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-S-indacene-propionic acid and the other fluorophore is selected from the group consisting of 6-carboxy-X-rhodamine, N,N,N′,N′-tetramethyl-6-carboxyrhodamine, Texas Red, Eosin, fluorescein, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid and 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-S-indacene-propionic acid.
Claim 4
Dependent Claim
19 words
The kit according to claim 2 wherein one fluorophore is fluorescein and the other fluorophore is 6-carboxy-X-rhodamine or N,N,N′,N′-tetramethyl-6-carboxyrhodamine.
Claim 5
Dependent Claim
86 words
A kit for detecting the presence of a target site of at least one nucleotide in a nucleic acid, the kit comprising: (a) a first polynucleotide covalently linked to a first fluorophore; and (b) a second polynucleotide covalently linked to a second fluorophore wherein the second polynucleotide is capable of being ligated to the first polynucleotide to form an oligonucleotide which binds to the target site, wherein the emission spectrum of one of the fluorophores overlaps the excitation spectrum of the other, packaged in a container.
Claim 6
Dependent Claim
86 words
A kit for detecting the presence of a target site of at least one nucleotide in a nucleic acid, the kit comprising: (a) a first polynucleotide covalently linked to a first fluorophore; and (b) a second polynucleotide covalently linked to a second fluorophore wherein the second polynucleotide is capable of being ligated to the first polynucleotide to form an oligonucleotide which binds to the target site, wherein the emission spectrum of one of the fluorophores overlaps the excitation spectrum of the other, packaged in a container.
Claim 7
Dependent Claim
51 words
The kit according to claim 3 wherein the first fluorophore is selected from the group consisting of fluorescein, cascade blue, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid and 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-S-indacene-propionic acid and the other fluorophore is selected from the group consisting of 6-carboxy-X-rhodamine, N,N,N′,N′-tetramethyl-6-carboxyrhodamine, Texas Red, Eosin, fluorescein, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid and 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-S-indacene-propionic acid.
Claim 8
Dependent Claim
18 words
The kit according to claim 5 wherein one fluorophore is fluorescein and the other fluorophore is Texas Red.
Claim 9
Dependent Claim
44 words
The kit of claim 1 , wherein the fluorophore that is linked to the polynucleotide is separated by at least about 20 nucleotides from the fluorophore that is linked to the dideoxynucleotide after the dideoxynucleotide has been added by 3′ extension to the polynucleotide.
Claim 10
Dependent Claim
44 words
The kit of claim 7 , wherein the fluorophore that is linked to the polynucleotide is separated by at least about 40 nucleotides from the fluorophore that is linked to the dideoxynucleotide after the dideoxynucleotide has been added by 3′ extension to the polynucleotide.
Claim 11
Dependent Claim
49 words
The kit of claim 4 wherein the first fluorophore that is linked to the first polynucleotide is separated by at least about 20 nucleotides from the second fluorophore that is linked to the second polynucleotide after the second polynucleotide has been added by 3′ extension to the first polynucleotide.
Claim 12
Dependent Claim
49 words
The kit of claim 9 wherein the first fluorophore that is linked to the first polynucleotide is separated by at least about 40 nucleotides from the second fluorophore that is linked to the second polynucleotide after the second polynucleotide has been added by 3′ extension to the first polynucleotide.
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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) | 2004-01-23 | 2004-07-24 | 2005-01-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2008-01-23 | 2008-07-24 | 2009-01-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2012-01-23 | 2012-07-24 | 2013-01-23 | $7700.00 | Window Closed / Lapsed |