US5691145A
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Detection of nucleic acids using G-quartets
Assignee: Becton Dickinson and Co
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Filing: 1996-08-27
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Granted: 1997-11-25
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Expires: 2016-08-27
Abstract
Abstract Oligonucleotides which form G-quartet structures have been found to be useful in fluorescence assays to detect a selected nucleic acid sequence. When one end of the oligonucleotide is labeled with a donor fluorophore and the other end is labeled with an acceptor dye, the folding of the molecule in the G-quartet structure brings the donor-acceptor pair into close proximity, allowing an interaction between the two labels which results in quenching of donor fluorescence or a change in other fluorescence properties which are the result of the interaction of two dyes in close proximity. The G-quartet structure unfolds upon hybridization to its complementary sequence, increasing the distance between the two dye labels. This results in decreased donor quenching or a change in another proximity-related fluorescence parameter. The associated increase in donor fluorescence intensity or the change in another fluorescence parameter may be monitored as an indication of the presence of a selected nucleic acid sequence. Alternatively, in some cases a decrease in acceptor fluorescence may be monitored as an indication of the presence of the selected nucleic acid sequence when the acceptor is also a fluorophore.
Inventors
- 👤 J. Bruce Pitner
- 👤 Glenn P. Vonk
- 👤 James G. Nadeau
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 09:01
CPC Classifications
G; G01; G01N; G01N33/00; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; Y; Y10; Y10S; Y10S436/00; Y10S436/80; Y10S436/805; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 26
Claim 1
Independent Claim
59 words
An oligonucleotide which forms an intramolecular G-quartet structure, the oligonucleotide being labeled with a donor fluorophore and an acceptor, the donor fluorophore and the acceptor selected such that fluorescence of the donor fluorophore is quenched by the acceptor when the oligonucleotide forms the G-quartet structure and quenching of donor fluorophore fluorescence is reduced upon unfolding of the G-quartet structure.
Claim 2
Dependent Claim
59 words
An oligonucleotide which forms an intramolecular G-quartet structure, the oligonucleotide being labeled with a donor fluorophore and an acceptor, the donor fluorophore and the acceptor selected such that fluorescence of the donor fluorophore is quenched by the acceptor when the oligonucleotide forms the G-quartet structure and quenching of donor fluorophore fluorescence is reduced upon unfolding of the G-quartet structure.
Claim 3
Dependent Claim
18 words
The oligonucleotide of claim 1 consisting of SEQ ID NO:2 labeled with the donor fluorophore and the acceptor.
Claim 4
Dependent Claim
18 words
The oligonucleotide of claim 1 consisting of SEQ ID NO:3 labeled with the donor fluorophore and the acceptor.
Claim 5
Dependent Claim
18 words
The oligonucleotide of claim 1 consisting of SEQ ID NO:4 labeled with the donor fluorophore and the acceptor.
Claim 6
Dependent Claim
18 words
The oligonucleotide of claim 1 consisting of SEQ ID NO:5 labeled with the donor fluorophore and the acceptor.
Claim 7
Dependent Claim
18 words
The oligonucleotide of claim 1 consisting of SEQ ID NO:7 labeled with the donor fluorophore and the acceptor.
Claim 8
Dependent Claim
37 words
The oligonucleotide of claim 1 wherein the donor fluorophore is fluorescein and the acceptor is selected from the group consisting of tetramethylrhodamine, Texas Red™, N-hydroxysuccinimidyl 1-pyrenebutyrate, eosin, N-hydroxysuccinimidyl 1-pyrenesulfonate (PYS), Rhodamine X, tetramethylrhodamine and p-(dimethylaminophenylazo) benzoic acid.
Claim 9
Dependent Claim
16 words
The oligonucleotide of claim 7 wherein the donor fluorophore is fluorescein and the acceptor is Rhodamine-X.
Claim 10
Independent Claim
130 words
A method for detecting or identifying a selected nucleic acid comprising: a) providing an oligonucleotide probe comprising an intramolecular G-quartet structure and a sequence complementary to the selected nucleic acid, the oligonucleotide being labeled with a donor fluorophore and an acceptor such that fluorescence of the donor fluorophore is quenched by the acceptor when the oligonucleotide forms the G-quartet structure, and quenching of donor fluorescence is reduced upon unfolding of the G-quartet structure; b) contacting the oligonucleotide probe with a nucleic acid sample, whereby if the selected nucleic acid is present the oligonucleotide probe binds to the selected nucleic acid and is rendered double-stranded, thereby unfolding the G-quartet structure and reducing quenching of donor fluorescence; and c) detecting a change in fluorescence; thereby detecting or identifying the selected nucleic acid.
Claim 11
Independent Claim
130 words
A method for detecting or identifying a selected nucleic acid comprising: a) providing an oligonucleotide probe comprising an intramolecular G-quartet structure and a sequence complementary to the selected nucleic acid, the oligonucleotide being labeled with a donor fluorophore and an acceptor such that fluorescence of the donor fluorophore is quenched by the acceptor when the oligonucleotide forms the G-quartet structure, and quenching of donor fluorescence is reduced upon unfolding of the G-quartet structure; b) contacting the oligonucleotide probe with a nucleic acid sample, whereby if the selected nucleic acid is present the oligonucleotide probe binds to the selected nucleic acid and is rendered double-stranded, thereby unfolding the G-quartet structure and reducing quenching of donor fluorescence; and c) detecting a change in fluorescence; thereby detecting or identifying the selected nucleic acid.
Claim 12
Dependent Claim
13 words
The method of claim 9, wherein an increase in donor fluorescence is detected.
Claim 13
Dependent Claim
19 words
The method of claim 9, wherein the acceptor is a fluorophore and a decrease in acceptor fluorescence is detected.
Claim 14
Dependent Claim
17 words
The method of claim 9 wherein the oligonucleotide is rendered double-stranded by hybridization to a complementary sequence.
Claim 15
Dependent Claim
22 words
The method of claim 9 wherein the oligonucleotide is rendered double-stranded by synthesis of its complement using the oligonucleotide as a template.
Claim 16
Dependent Claim
21 words
The method of claim 9 wherein the oligonucleotide consists of SEQ ID NO:2 labeled with the donor fluorophore and the acceptor.
Claim 17
Dependent Claim
21 words
The method of claim 9 wherein the oligonucleotide consists of SEQ ID NO:3 labeled with the donor fluorophore and the acceptor.
Claim 18
Dependent Claim
21 words
The method of claim 9 wherein the oligonucleotide consists of SEQ ID NO:4 labeled with the donor fluorophore and the acceptor.
Claim 19
Dependent Claim
21 words
The method of claim 9 wherein the oligonucleotide consists of SEQ ID NO:5 labeled with the donor fluorophore and the acceptor.
Claim 20
Dependent Claim
21 words
The method of claim 9 wherein the oligonucleotide consists of SEQ ID NO:7 labeled with the donor fluorophore and the acceptor.
Claim 21
Dependent Claim
37 words
The method of claim 9 wherein the donor fluorophore is fluorescein and the acceptor is selected from the group consisting of tetramethylrhodamine, Texas Red™, N-hydroxysuccinimidyl 1-pyrenebutyrate, eosin, N-hydroxysuccinimidyl 1-pyrenesulfonate (PYS), Rhodamine X, tetramethylrhodamine and p-(dimethylaminophenylazo) benzoic acid.
Claim 22
Dependent Claim
24 words
The method of claim 9 wherein the nucleic acid sequence to be detected or identified is a product of a nucleic acid amplification reaction.
Claim 23
Dependent Claim
21 words
The method of claim 20 wherein the nucleic acid sequence to be detected is the amplification product of an SDA reaction.
Claim 24
Independent Claim
132 words
A method for detecting or identifying a selected nucleic acid comprising: a) providing an oligonucleotide probe comprising an intramolecular G-quartet structure and a sequence complementary to the selected nucleic acid, the oligonucleotide being labeled with a donor fluorophore and an acceptor such that unfolding of the G-quartet structure increases the distance between the donor fluorophore and acceptor; b) contacting the oligonucleotide probe with a nucleic acid sample, whereby if the selected nucleic acid is present the oligonucleotide probe binds to the selected nucleic acid and is rendered double-stranded, thereby unfolding the G-quartet structure and increasing the distance between the donor fluorophore and acceptor; and c) detecting a change in a fluorescence property associated with the increased distance between the donor fluorophore and the acceptor, thereby detecting or identifying the selected nucleic acid.
Claim 25
Independent Claim
132 words
A method for detecting or identifying a selected nucleic acid comprising: a) providing an oligonucleotide probe comprising an intramolecular G-quartet structure and a sequence complementary to the selected nucleic acid, the oligonucleotide being labeled with a donor fluorophore and an acceptor such that unfolding of the G-quartet structure increases the distance between the donor fluorophore and acceptor; b) contacting the oligonucleotide probe with a nucleic acid sample, whereby if the selected nucleic acid is present the oligonucleotide probe binds to the selected nucleic acid and is rendered double-stranded, thereby unfolding the G-quartet structure and increasing the distance between the donor fluorophore and acceptor; and c) detecting a change in a fluorescence property associated with the increased distance between the donor fluorophore and the acceptor, thereby detecting or identifying the selected nucleic acid.
Claim 26
Dependent Claim
25 words
The method of claim 22 wherein the fluorescence property is selected from the group consisting of fluorescence intensity, fluorescence lifetime, fluorescence polarization and fluorescence anisotropy.
References Cited (Backward Citations)
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