Detection of nucleic acids by fluorescence quenching

• US 5,935,791 A
• Filed: 09/23/1997
• Issued: 08/10/1999
• Est. Priority Date: 09/23/1997
• Status: Expired due to Fees

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Pitner, J. Bruce, Nadeau, James G., Hsieh, Helen V., Linn, C. Preston
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/933,749
• Time in Patent Office: 686 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/24.3, 536/24.33
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid

Abstract

Detector nucleic acids are employed for detection of nucleic acid target sequences by fluorescence quenching mechanisms. The detector nucleic acid comprises at least two oligonucleotides and is partially single-stranded and partially double-stranded. One of the two dyes of a donor/acceptor dye pair is linked to the first oligonucleotide and the other is linked to a second oligonucleotide such that they are in close spatial proximity when the first and second oligonucleotides are base-paired and donor fluorescence is quenched. A single second oligonucleotide may be hybridized to the first oligonucleotide or multiple second oligonucleotides may be hybridized to the first oligonucleotide and to each other, forming a junction structure comprising multiple donor/acceptor dye pairs. The detector oligonucleotide retains its partially single-stranded and partially double-stranded conformation in the absence of target. In the presence of target, however, the second oligonucleotide(s) of the detector nucleic acid is/are completely or partially displaced from the first, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence which may be detected as an indication of the presence of the target sequence.

Claims

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

a) hybridizing to the target sequence a detector nucleic acid comprising

i) a first oligonucleotide hybridized to a second oligonucleotide which is shorter than the first oligonucleotide such that the detector nucleic acid comprises an intermolecularly base-paired portion and a single-stranded target binding sequence, and;

ii) a first dye linked to the first oligonucleotide and a second dye linked to the second oligonucleotide which together form a fluorescent donor/acceptor dye pair linked to the detector nucleic acid such that fluorescence of the first or the second dye is quenched;

b) extending the target sequence on the first oligonucleotide, thereby displacing the second oligonucleotide from the first oligonucleotide and producing a change in a fluorescence parameter, and;

c) detecting the change in the fluorescence parameter as an indication of the presence of the target sequence.

2. The method of claim 1 wherein a change in fluorescence intensity is detected as an indication of the presence of the target sequence.

3. The method of claim 2 wherein an increase in donor dye fluorescence intensity or a decrease in acceptor dye fluorescence intensity is detected as an indication of the presence of the target sequence.

4. The method of claim 2 wherein the change in fluorescence intensity is detected as a) an increase in a ratio of donor dye fluorescence after displacing the second oligonucleotide to donor dye fluorescence prior to displacing the second oligonucleotide, or b) as a decrease in a ratio of acceptor dye fluorescence after displacing the second oligonucleotide to acceptor dye fluorescence prior to displacing the second oligonucleotide.

5. The method of claim 1 wherein a change in fluorescence lifetime is detected as an indication of the presence of the target sequence.

6. The method of claim 1 wherein the change in the fluorescence parameter is detected in real-time.

7. The method of claim 1 wherein the change in the fluorescence parameter is detected at an endpoint.

8. The method of claim 1 wherein the fluorescent donor/acceptor dye pair comprises fluorescein and Rhodamine X, Rhodamine X and Cy5, or fluorescein and Dabcyl.

9. The method of claim 1 wherein the intermolecularly base-paired portion of the detector nucleic acid comprises a portion of the target binding sequence.

10. The method of claim 1 wherein multiple second oligonucleotides are hybridized to the first oligonucleotide and to each other such that the intermolecularly base-paired portion of the detector nucleic acid forms an oligonucleotide junction structure.

11. The method of claim 10 wherein the oligonucleotide junction structure is a 3-way, 4-way, 5-way or 6-way junction.

12. A method for detecting amplification of a target sequence comprising, in an amplification reaction:

a) hybridizing to the target sequence a detector nucleic acid comprising

i) a first oligonucleotide hybridized to a second oligonucleotide which is shorter than the first oligonucleotide such that the detector nucleic acid comprises an intermolecularly base-paired portion and a single-stranded target binding sequence, and;

ii) a first dye linked to the first oligonucleotide and a second dye linked to the second oligonucleotide which together form a fluorescent donor/acceptor dye pair linked to the detector nucleic acid such that fluorescence of the first or the second dye is quenched;

b) extending the first oligonucleotide of the detector nucleic acid on the target sequence with a polymerase to produce a detector nucleic acid extension product and separating the detector nucleic acid extension product from the target sequence;

c) hybridizing a primer to the extended first oligonucleotide of the detector nucleic acid extension product and extending the primer with the polymerase, thereby displacing the second oligonucleotide from the extended first oligonucleotide and producing a change in a fluorescence parameter, and;

d) detecting the change in the fluorescence parameter as an indication of amplification of the target sequence.

13. The method of claim 12 wherein the target sequence is amplified by Strand Displacement Amplification.

14. The method of claim 12 wherein the target sequence is amplified by the Polymerase Chain Reaction.

15. The method of claim 12 wherein the target sequence is amplified by 3SR, TMA or NASBA.

16. The method of claim 12 wherein a change in fluorescence intensity is detected.

17. The method of claim 16 wherein an increase in fluorescence intensity of the first or the second dye, or a decrease in fluorescence intensity of the first or the second dye is detected as an indication of amplification of the target sequence.

18. The method of claim 16 wherein the change in fluorescence intensity is detected as a) an increase in a ratio of donor dye fluorescence after displacing the second oligonucleotide and donor dye fluorescence prior to displacing the second oligonucleotide, or b) as a decrease in a ratio of acceptor dye fluorescence after displacing the second oligonucleotide and acceptor dye fluorescence prior to displacing the second oligonucleotide.

19. The method of claim 16 wherein the change in fluorescence intensity is detected in real-time.

20. The method of claim 16 wherein the change in fluorescence intensity is detected at a selected end-point in the amplification reaction.

21. The method of claim 12 wherein the fluorescent donor/acceptor dye pair comprises fluorescein and Rhodamine X, Rhodamine X and Cy5, or fluorescein and Dabcyl.

22. The method of claim 12 wherein the intermolecularly base-paired portion of the detector nucleic acid comprises a portion of the target binding sequence.

23. The method of claim 12 wherein multiple second oligonucleotides are hybridized to the first oligonucleotide and to each other such that the intermolecularly base-paired portion of the detector nucleic acid forms an oligonucleotide junction structure.

24. The method of claim 23 wherein the oligonucleotide junction structure is a 3-way, 4-way, 5-way or 6-way junction.

45. The method of claim 12 wherein the detector nucleic acid is an amplification primer.

46. The method of claim 12 wherein the detector nucleic acid is a signal primer.

47. The method of claim 12 wherein the primer is an amplification primer.

25. A method for detecting a target sequence comprising:

a) providing a detector nucleic acid comprising

i) a first oligonucleotide hybridized to a second oligonucleotide which is shorter than the first oligonucleotide such that the detector nucleic acid comprises an intermolecularly base-paired portion and a single-stranded target binding sequence, and;

ii) a first dye linked to the first oligonucleotide and a second dye linked to the second oligonucleotide which together form a fluorescent donor/acceptor dye pair linked to the detector nucleic acid such that fluorescence of the first or the second dye is quenched;

b) hybridizing the detector nucleic acid to the target sequence by means of the single-stranded target binding sequence, thereby reducing quenching of the first or the second dye and producing a change in a fluorescence parameter, and;

c) detecting the change in the fluorescence parameter as an indication of the presence of the target sequence.

26. The method of claim 25 wherein the intermolecularly base-paired portion of the detector nucleic acid comprises a portion of the target binding sequence.

27. The method of claim 25 wherein the intermolecularly base-paired portion of the detector nucleic acid is 3'"'"' to the single-stranded target binding sequence.

28. The method of claim 25 wherein the intermolecularly base-paired portion of the detector nucleic acid is 5'"'"' to the single-stranded target binding sequence.

29. The method of claim 25 wherein the change in the fluorescence parameter is detected in real-time.

30. The method of claim 25 wherein the change in the fluorescence parameter is detected at an endpoint.

31. The method of claim 25 wherein the change in the fluorescence parameter is a change in fluorescence intensity.

32. The method of claim 25 wherein the detector nucleic acid is immobilized on a solid phase.

33. The method of claim 25 wherein multiple second oligonucleotides are hybridized to the first oligonucleotide and to each other such that the intermolecularly base-paired portion of the detector nucleic acid forms an oligonucleotide junction structure.

34. The method of claim 33 wherein the oligonucleotide junction structure is a 3-way, 4-way, 5-way or 6-way junction.

35. A detector nucleic acid comprising:

a) a first oligonucleotide hybridized to a second oligonucleotide which is shorter than the first oligonucleotide such that the detector nucleic acid comprises an intermolecularly base-paired portion and single-stranded target binding sequence, wherein at least a portion of the intermolecularly base-paired portion is not complementary to a selected target, and;

b) a first dye linked to the first oligonucleotide and a second dye linked to the second oligonucleotide which together form a fluorescent donor/acceptor dye pair linked to the detector nucleic acid such that fluorescence of the first or the second dye is quenched and dissociation of the detector nucleic acid reduces quenching of the first or second dye.

36. The detector nucleic acid of claim 35 wherein the intermolecularly base-paired portion of the detector nucleic acid is 5'"'"' to the single-stranded target binding sequence.

37. The detector nucleic acid of claim 36 wherein the intermolecularly base-paired portion of the detector nucleic acid comprises a portion of the target binding sequence.

38. The detector nucleic acid of claim 35 wherein the intermolecularly base-paired portion of the detector nucleic acid is 3'"'"' to the target binding sequence.

39. The detector nucleic acid of claim 38 wherein the intermolecularly base-paired portion of the detector nucleic acid comprises a portion of the target binding sequence.

40. The detector nucleic acid of claim 35 wherein the fluorescent donor/acceptor dye pair comprises fluorescein and Rhodamine X, Cy5 and Rhodamine X, or fluorescein and Dabcyl.

41. The detector nucleic acid of claim 35 which is immobilized on a solid phase.

42. The detector nucleic acid of claim 35 wherein multiple second oligonucleotides are hybridized to the first oligonucleotide and to each other such that the intermolecularly base-paired portion of the detector nucleic acid forms an oligonucleotide junction structure.

43. The detector nucleic acid of claim 42 wherein the oligonucleotide junction structure is a 3-way, 4-way, 5-way or 6-way junction.

44. The detector nucleic acid of claim 42 which comprises multiple fluorescent donor/acceptor dye pairs.