Fluorerscence polarization detection of nucleic acid amplication

• US 5,593,867 A
• Filed: 09/23/1994
• Issued: 01/14/1997
• Est. Priority Date: 04/18/1994
• Status: Expired due to Fees

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Nadeau, James G., Walker, G. Terrance, Linn, C. Preston
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/311,474
• Time in Patent Office: 844 Days
• Field of Search: 435/6, 435/91.2, 435/91, 435/94, 935/78
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded

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.

Claims

1. 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.

2. The method of claim 1 wherein the detector probe is present in the SDA reaction at 50 pm-10 nM.

3. The method of claim 2 wherein the detector probe is labeled with La Jolla Blue™

or fluorescein.

4. 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.

5. The method of claim 1 wherein the increase in fluorescence polarization is detected during amplification by transient state fluorescence polarization.

6. The method of claim 1 wherein the increase in fluorescence polarization is detected after target amplification by steady state or transient state fluorescence polarization.

7. The method of claim 1 further comprising quantitating the initial concentration of the target sequence from the increase in fluorescence polarization.

8. The method of claim 1 wherein the target sequence is an M. tuberculosis target sequence.

13. 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.

17. 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.

18. The method of claim 17 wherein the detector probe comprises a recognition site for EcoRI and the SDA reaction further comprises EcoRI (Gln 111).

9. 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.

10. The method of claim 9 wherein the amplification reaction is SDA.

11. The method of claim 9 wherein the amplification reaction is PCR.

12. The method of claim 9 wherein the amplification reaction is 3SR.

14. 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.

15. 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.

16. The method of claim 9 wherein the amplification reaction further comprises an oligonucleotide which hybridizes to the double-stranded detector probe extension product.