Fluorescence polarization detection of nucleic acids

• US 5,641,633 A
• Filed: 11/15/1995
• Issued: 06/24/1997
• Est. Priority Date: 11/15/1995
• Status: Expired due to Fees

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Walker, G. Terrance, Spears, Patricia Anne, Linn, Carl Preston
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/559,508
• Time in Patent Office: 587 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.53, 536/24.3, 536/24.33, 536/25.32, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation

Abstract

The present invention provides methods for detecting amplified or unamplified nucleic acid target sequences at increased temperatures by changes in fluorescence polarization. The decrease in fluorescence polarization associated with hybridization of oligonucleotides at higher, more stringent, temperatures is overcome by including a double-stranded DNA binding protein in the assay. At elevated temperatures, the double-stranded DNA binding protein restores, and often enhances, the magnitude of the change in fluorescence polarization associated with single- to double-stranded conversion of an oligonucleotide probe or primer.

Claims

1. A method for detecting a target nucleic acid sequence comprising:

a) converting a single-stranded oligonucleotide comprising a fluorescent label to double-stranded form by hybridizing the single-stranded oligonucleotide to the target sequence at about 45°

-75°

C.

b) binding a double-stranded DNA binding protein to the double-stranded form at about 45°

-75°

; and

c) detecting the target sequence by detecting a change in fluorescence polarization resulting from conversion of the single-stranded oligonucleotide to the double-stranded form with the double-stranded DNA binding protein bound thereto at about 45 °

-75°

C.

2. The method of claim 1 wherein the single-stranded oligonucleotide is a probe which is converted to double-stranded form upon hybridization to the target sequence.

3. The method of claim 2 wherein the hybridized probe is extended on the target sequence prior to detecting the change in fluorescence polarization.

4. The method of claim 1 wherein the single-stranded oligonucleotide is hybridized to the target sequence at about 50°

-60°

C.

5. The method of claim 1 wherein the double-stranded DNA binding protein binds to a specific recognition sequence in the double-stranded form of the oligonucleotide.

6. The method of claim 5 wherein the double-stranded DNA binding protein is a restriction endonuclease.

7. The method of claim 1 wherein the double-stranded DNA binding protein binds sequence non-specifically to the double-stranded form of the oligonucleotide.

8. The method of claim 7 wherein the double-stranded DNA binding protein is a polymerase.

9. The method of claim 1 wherein the fluorescent label is 5-(4,6-dichlorotriazin-2-yl) amino fluorescein).

10. The method of claim 1 wherein the change in fluorescence polarization is detected in real-time as the single-stranded oligonucleotide is converted to double-stranded form.

11. The method of claim 1 wherein the change in fluorescence polarization is detected as an endpoint measurement upon completion of conversion of the single-stranded oligonucleotide to double-stranded form.

12. A method for detecting amplification of a target nucleic acid sequence comprising:

a) amplifying the target sequence at about 45°

-75°

C. such that a single-stranded oligonucleotide primer comprising a fluorescent label is converted to double-stranded form as a result of target sequence amplification;

b) binding a double-stranded DNA binding protein to the double-stranded form at about 45°

-75°

C.; and

c) detecting target sequence amplification by detecting a change in fluorescence polarization resulting from conversion of the single-stranded primer to the double-stranded form with the double-stranded DNA binding protein bound thereto at about 45°

-75°

C.

21. The method of claim 12 wherein the single-stranded primer is an amplification primer.

13. The method of 12 wherein the change in fluorescence polarization is detected in real-time as the single-stranded primer is converted to double-stranded form.

14. The method of 12 wherein the change in fluorescence polarization is detected as an endpoint measurement upon termination of target sequence amplification.

15. The method of 12 wherein the single-stranded primer is a signal primer.

16. The method of 12 wherein the double-stranded DNA binding protein binds to a specific recognition sequence in the double-stranded form of the primer.

17. The method of 16 wherein the double-stranded DNA binding protein is a restriction endonuclease.

18. The method of 12 wherein the double-stranded DNA binding protein binds sequence non-specifically to the double-stranded form of the primer.

19. The method of 18 wherein the double-stranded DNA binding protein is a polymerase.

22. The method of claim 19 wherein the polymerase is a polymerase used to amplify the target sequence.

23. The method of claim 22 wherein the single-stranded oligonucleotide primer is a signal primer which is converted to double-stranded form in a target amplification-dependent manner.

24. The method of claim 22 wherein a second double-stranded DNA binding protein is bound to the double-stranded form prior to detecting the change in fluorescence polarization.

25. The method of claim 22 wherein the target sequence is amplified by thermophilic Strand Displacement Amplification.

26. The method of claim 22 wherein the target sequence is amplified by the Polymerase Chain Reaction.

20. The method of 12 wherein the fluorescent label is 5-(4,6-dichlorotriazin-2-yl) amino fluorescein).