US5945283A
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US
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Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer
Assignee: Washington University in St. Louis
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Filing: 1996-12-17
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Granted: 1999-08-31
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Expires: 2016-12-17
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
2026-11-06 02:04
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C12Q2600/00; C12Q2600/156
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
106 words
A method for detecting presence of a target site of at least one nucleotide in a sequence of contiguous nucleotides in a sample of nucleic acid comprising: (a) synthesizing, in the sample, an oligonucleotide on and directed by a template of the contiguous nucleotides including the target site wherein the oligonucleotide comprises two fluorophores each of which is covalently linked to a separate nucleotide; and (b) detecting a fluorescence energy transfer from one fluorophore to the other upon denaturation and release of the oligonucleotide from the contiguous nucleotides including the target site wherein the detecting of fluorescence energy transfer indicates the presence of the target site.
Claim 2
Independent Claim
106 words
A method for detecting presence of a target site of at least one nucleotide in a sequence of contiguous nucleotides in a sample of nucleic acid comprising: (a) synthesizing, in the sample, an oligonucleotide on and directed by a template of the contiguous nucleotides including the target site wherein the oligonucleotide comprises two fluorophores each of which is covalently linked to a separate nucleotide; and (b) detecting a fluorescence energy transfer from one fluorophore to the other upon denaturation and release of the oligonucleotide from the contiguous nucleotides including the target site wherein the detecting of fluorescence energy transfer indicates the presence of the target site.
Claim 3
Dependent Claim
53 words
The method according to claim 1 wherein the synthesizing step comprises template-directed 3' extension of a polynucleotide, which is covalently linked to one fluorophore and bound either to the target site or immediately 3' to the target site on the template, by addition of a dideoxynucleoside triphosphate covalently linked to the other fluorophore.
Claim 4
Dependent Claim
51 words
The method according to claim 2 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,4-adiaza-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 5
Dependent Claim
19 words
The method according to claim 3 wherein one fluorophore is fluorescein and the other fluorophore is 6-carboxy-X-rhodamine or N,N,N',N'-tetramethyl-6-carboxyrhodamine.
Claim 6
Dependent Claim
31 words
The method according to claim 3 wherein detecting an increase in emission of one fluorophore upon excitation of the other indicates the presence of the target site in the nucleic acid.
Claim 7
Dependent Claim
24 words
The method according to claim 3 wherein detecting a quenching of one fluorophore indicates the presence of the target site in the nucleic acid.
Claim 8
Dependent Claim
13 words
The method according to claim 3 wherein the method detects single nucleotide polymorphism.
Claim 9
Dependent Claim
44 words
The method according to claim 1 wherein the synthesizing step comprises a template-directed ligation of a first polynucleotide, which is covalently linked to one fluorophore and bound to the target site on the template, to a second polynucleotide covalently linked to the other fluorophore.
Claim 10
Dependent Claim
51 words
The method according to claim 8 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,4-adiaza-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,4-adiaza-S-indacene-propionic acid.
Claim 11
Dependent Claim
19 words
The method according to claim 9 wherein one fluorophore is fluorescein and the other fluorophore is 6-carboxy-X-rhodamine or N,N,N',N'-tetramethyl-6-carboxyrhodamine.
Claim 12
Dependent Claim
31 words
The method according to claim 9 wherein detecting an increase in emission of one fluorophore upon excitation of the other indicates the presence of the target site in the nucleic acid.
Claim 13
Dependent Claim
24 words
The method according to claim 9 wherein detecting a quenching of one fluorophore indicates the presence of the target site in the nucleic acid.
Claim 14
Dependent Claim
13 words
The method according to claim 9 wherein the method detects single nucleotide polymorphism.
Claim 15
Independent Claim
117 words
A method for detecting presence of a target site of at least one nucleotide in a sequence of contiguous nucleotides in a sample of nucleic acid comprising: (a) performing, in the sample, a reaction which synthesizes an oligonucleotide on and directed by a template of the contiguous nucleotides including the target site if the target site is present in said sample, wherein the oligonucleotide comprises two fluorophores each of which is covalently linked to a separate nucleotide; and (b) detecting a fluorescence energy transfer from one fluorophore to the other upon denaturation and release of oligonucleotide if it is synthesized in step (a), wherein the detecting of fluorescence energy transfer indicates the presence of the target site.
Claim 16
Independent Claim
117 words
A method for detecting presence of a target site of at least one nucleotide in a sequence of contiguous nucleotides in a sample of nucleic acid comprising: (a) performing, in the sample, a reaction which synthesizes an oligonucleotide on and directed by a template of the contiguous nucleotides including the target site if the target site is present in said sample, wherein the oligonucleotide comprises two fluorophores each of which is covalently linked to a separate nucleotide; and (b) detecting a fluorescence energy transfer from one fluorophore to the other upon denaturation and release of oligonucleotide if it is synthesized in step (a), wherein the detecting of fluorescence energy transfer indicates the presence of the target site.
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