EP0678581A1
Expired / Lapsed
EP
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Fluorescence polarization detection of nucleic acid amplification
Assignee: Becton Dickinson and Co
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Filing: 1995-04-03
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Granted: —
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Expires: 2015-04-03
Abstract
Abstract Fluorescence polarization methods for detection of nucleic acid amplification. A fluorescently labelled oligodeoxynucleotide probe is converted from single- to double-stranded form in a target dependent manner during amplification of the target sequence. This conformational change is accompanied by an increase in fluorescence polarization values. The iincrease in fluorescence polarization can be measured on a transient-state fluorometer in real-time during the amplification reaction without any physical manipulation of the sample. The inventive methods therefore provide a closed, homogeneous system for both amplification of target sequences and detection of amplification. Alternatively, amplification may be detected in the fluorometer after the amplification reaction is completed.
Inventors
- 👤 George Terrance Walker
- 👤 James Gregory Nadeau
- 👤 Carl Preston Linn
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-01 16:48
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6851
Claims Analysis
Total Claims: 10
Claim 1
Independent Claim
168 words
A method for detecting amplification of a double-stranded nucleic acid target sequence in a Strand Displacement Amplification reaction (SDA), the method comprising: a) including in the SDA reaction a first amplification primer for SDA which hybridizes to a first strand of the target sequence, and a fluorescently-labeled single-stranded detector probe which hybridizes to the first strand of the target sequence downstream of the first amplification primer; b) extending the first amplification primer and the detector probe on the target sequence, thereby (i) producing a detector probe extension product which is displaced from the first strand of the target sequence by extension of the first amplification primer and (ii) amplifying the target sequence; c) hybridizing to the extension product a second amplification primer for SDA which hybridizes to a second strand of the target sequence complementary to the first strand of the target sequence; d) extending the second amplification primer, thereby converting the detector probe extension product to double-stranded form, and; e) detecting target amplification by measuring fluorescence polarization.
Claim 2
Dependent Claim
17 words
The method of Claim 1 wherein the detector probe is labeled with La Jolla Blue or fluorescein.
Claim 3
Dependent Claim
17 words
The method of Claim 1 wherein fluorescence polarization is measured during amplification by transient state fluorescence polarization.
Claim 4
Dependent Claim
21 words
The method of Claim 1 wherein fluorescence polarization is measured after target amplification by steady state or transient state fluorescence polarization.
Claim 5
Dependent Claim
20 words
The method of Claim 1 further comprising quantitating the initial concentration of the target sequence from the measured fluorescence polarization.
Claim 6
Dependent Claim
19 words
The method of Claim 1 wherein the detector probe comprises a recognition site for a nucleic acid binding protein.
Claim 7
Independent Claim
161 words
A method for detecting amplification of a nucleic acid target sequence in an amplification reaction, the method comprising: a) including in the amplification reaction a first amplification primer which hybridizes to a first strand of the target sequence and a fluorescently-labeled detector probe which hybridizes to the first strand of the target sequence downstream of the first amplification primer; b) extending the first amplification primer and the detector probe on the target sequence, thereby (i) producing a detector probe extension product which is displaced from the first strand of the target sequence by extension of the first amplification primer and (ii) amplifying the target sequence: c) hybridizing to the detector probe extension product a second amplification primer which hybridizes to a second strand of the target sequence complementary to the first strand of the target sequence; d) extending the second amplification primer, thereby converting the detector probe extension product to double-stranded form, and; e) detecting target amplification by measuring fluorescence polarization.
Claim 8
Dependent Claim
11 words
The method of Claim 7 wherein the amplification reaction is SDA.
Claim 9
Dependent Claim
11 words
The method of Claim 7 wherein the amplification reaction is PCR.
Claim 10
Dependent Claim
11 words
The method of Claim 7 wherein the amplification reaction is 3SR.
References Cited (Backward Citations)
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