US5593867A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Fluorerscence polarization detection of nucleic acid amplication
Assignee: Becton Dickinson and Co
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Filing: 1994-09-23
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Granted: 1997-01-14
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Expires: 2014-09-23
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 increase 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
- 👤 G. Terrance Walker
- 👤 James G. Nadeau
- 👤 C. Preston Linn
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 17:15
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
186 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 producing (i) 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) a first amplification primer extension product; c) hybridizing a second amplification primer for SDA to the detector probe extension product and the first amplification primer extension product; d) extending the second amplification primer on the detector probe extension product and the first amplification primer extension product, thereby converting the detector probe extension product to double-stranded form and amplifying the target sequence, and; e) detecting an increase in fluorescence polarization as an indication of amplification of the target sequence.
Claim 2
Independent Claim
186 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 producing (i) 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) a first amplification primer extension product; c) hybridizing a second amplification primer for SDA to the detector probe extension product and the first amplification primer extension product; d) extending the second amplification primer on the detector probe extension product and the first amplification primer extension product, thereby converting the detector probe extension product to double-stranded form and amplifying the target sequence, and; e) detecting an increase in fluorescence polarization as an indication of amplification of the target sequence.
Claim 3
Dependent Claim
19 words
The method of claim 1 wherein the detector probe is present in the SDA reaction at 50 pm-10 nM.
Claim 4
Dependent Claim
17 words
The method of claim 2 wherein the detector probe is labeled with La Jolla Blue™ or fluorescein.
Claim 5
Dependent Claim
24 words
The method of claim 2 wherein the the SDA reaction comprises about 0.25 units of polymerase and 100 nM of the first amplification primer.
Claim 6
Dependent Claim
20 words
The method of claim 1 wherein the increase in fluorescence polarization is detected during amplification by transient state fluorescence polarization.
Claim 7
Dependent Claim
24 words
The method of claim 1 wherein the increase in fluorescence polarization is detected after target amplification by steady state or transient state fluorescence polarization.
Claim 8
Dependent Claim
21 words
The method of claim 1 further comprising quantitating the initial concentration of the target sequence from the increase in fluorescence polarization.
Claim 9
Dependent Claim
15 words
The method of claim 1 wherein the target sequence is an M. tuberculosis target sequence.
Claim 10
Independent Claim
175 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 producing (i) 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) a first amplification primer extension product; c) hybridizing a second amplification primer to the detector probe extension product and the first amplification primer extension product; d) extending the second amplification primer on the detector probe extension product and the first amplification primer extension product, thereby converting the detector probe extension product to double-stranded form and amplifying the target sequence, and; e) detecting an increase in fluorescence polarization as an indication of target sequence amplification.
Claim 11
Independent Claim
175 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 producing (i) 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) a first amplification primer extension product; c) hybridizing a second amplification primer to the detector probe extension product and the first amplification primer extension product; d) extending the second amplification primer on the detector probe extension product and the first amplification primer extension product, thereby converting the detector probe extension product to double-stranded form and amplifying the target sequence, and; e) detecting an increase in fluorescence polarization as an indication of target sequence amplification.
Claim 12
Dependent Claim
11 words
The method of claim 9 wherein the amplification reaction is SDA.
Claim 13
Dependent Claim
11 words
The method of claim 9 wherein the amplification reaction is PCR.
Claim 14
Dependent Claim
11 words
The method of claim 9 wherein the amplification reaction is 3SR.
Claim 15
Dependent Claim
25 words
The method of claim 1 wherein the SDA reaction further comprises a double-stranded DNA binding protein which binds to the double-stranded detector probe extension product.
Claim 16
Dependent Claim
25 words
The method of claim 9 wherein the amplification reaction further comprises a double-stranded DNA binding protein which binds to the double-stranded detector probe extension product.
Claim 17
Dependent Claim
37 words
The method of claim 14 wherein the detector probe comprises a recognition site for a double-stranded nucleic acid binding protein, and the amplification reaction further comprises the nucleic acid binding protein which binds to the recognition site.
Claim 18
Dependent Claim
22 words
The method of claim 9 wherein the amplification reaction further comprises an oligonucleotide which hybridizes to the double-stranded detector probe extension product.
Claim 19
Dependent Claim
37 words
The method of claim 13 wherein the detector probe comprises a recognition site for a double-stranded nucleic acid binding protein, and the SDA reaction further comprises the nucleic acid binding protein which binds to the recognition site.
Claim 20
Dependent Claim
24 words
The method of claim 17 wherein the detector probe comprises a recognition site for EcoRI and the SDA reaction further comprises EcoRI (Gln 111).
References Cited (Backward Citations)
WO1990006374A1
Cited
EP0382433A2
Cited
EP0500224A2
Cited
WO1992018650A1
Cited
EP0512334A2
Cited
WO1993009250A1
Cited
WO1993010267A1
Cited
US5270184A
Cited
WO1993016194A1
Cited
WO1994012665A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2000-01-14 | 2000-07-15 | 2001-01-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-01-14 | 2004-07-15 | 2005-01-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-01-14 | 2008-07-15 | 2009-01-14 | $7700.00 | Window Closed / Lapsed |