Detection of nucleic acids by fluorescence quenching

• US 5,846,726 A
• Filed: 05/13/1997
• Issued: 12/08/1998
• Est. Priority Date: 05/13/1997
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Pitner, J. Bruce, Schram, James L., Nadeau, James G., Walker, G. Terrance, Vonk, Glenn P., Linn, C. Preston
• Primary Examiner(s): LeGuyader, John L.
• Assistant Examiner(s): SHIBUYA, MARK LANCE
• Application Number:
  US08/855,085
• Time in Patent Office: 574 Days
• Field of Search: 435/6, 435/91.2, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.

Claims

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

a) hybridizing to the target sequence a signal primer comprising a target binding sequence and a single-stranded restriction endonuclease recognition sequence 5'"'"' to the target binding sequence, the restriction endonuclease recognition sequence flanked by a donor fluorophore and an acceptor dye such that fluorescence of the donor fluorophore is quenched;

b) in a primer extension reaction, synthesizing a complementary strand using the signal primer as a template, thereby rendering the restriction endonuclease recognition sequence double-stranded;

c) cleaving or nicking the double-stranded restriction endonuclease recognition sequence with a restriction endonuclease, thereby reducing donor fluorophore quenching and producing a change in a fluorescence parameter, and;

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

2. The method of claim 1 wherein the complementary strand is synthesized in a target amplification reaction.

3. The method of claim 1 wherein the complementary strand is synthesized by extension of the target sequence using the signal primer as a template.

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

5. The method of claim 4 wherein an increase in donor fluorophore fluorescence intensity or a decrease in acceptor dye fluorescence intensity is detected as an indication of the presence of the target sequence.

6. The method of claim 5 wherein the change in fluorescence intensity is detected as i) an increase in a ratio of donor fluorophore fluorescence after cleaving or nicking to donor fluorophore fluorescence in the signal primer, or ii) as a decrease in a ratio of acceptor dye fluorescence after cleaving or nicking to acceptor dye fluorescence in the signal primer.

7. The method of claim 1 wherein a change in fluorescence lifetime of the donor fluorophore or the acceptor dye is detected as an indication of the presence of the target sequence.

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

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

10. The method of claim 1 wherein the donor fluorophore and the acceptor dye are separated by about 8-20 nucleotides in the signal primer.

11. The method of claim 10 wherein the donor fluorophore and the acceptor dye are separated by about 10-16 nucleotides in the signal primer.

12. The method of claim 1 wherein the donor fluorophore is fluorescein and the acceptor dye is Rhodamine X.

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

a) hybridizing to the target sequence a first primer comprising a target binding sequence and a restriction endonuclease recognition sequence 5'"'"' to the target binding sequence, the restriction endonuclease recognition sequence flanked by a donor fluorophore and an acceptor dye such that fluorescence of the donor fluorophore is quenched;

b) extending the hybridized first primer on the target sequence with a polymerase to produce a first primer extension product and separating the first primer extension product from the target sequence;

c) rendering the separated first primer extension product and the restriction endonuclease recognition sequence double-stranded by hybridization and extension of a second primer;

d) cleaving or nicking the double-stranded restriction endonuclease recognition sequence with a restriction endonuclease, thereby reducing donor fluorophore quenching and producing a change in a fluorescence parameter, and;

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

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

15. The method of claim 14 further comprising nicking of the double-stranded restriction endonuclease recognition sequence in an amplification primer during target amplification.

16. The method of claim 15 wherein the restriction endonuclease is BsoBI or Aval.

17. The method of claim 14 further comprising a second restriction endonuclease which nicks a second double-stranded restriction endonuclease recognition sequence in an amplification primer during target amplification.

18. The method of claim 13 wherein the target sequence is amplified by the Polymerase Chain Reaction, 3SR, TMA or NASBA.

19. The method of claim 13 wherein the first primer is a signal primer.

20. The method of claim 19 wherein the second primer is an amplification primer.

21. The method of claim 13 wherein the first and second primers are amplification primers.

22. The method of claim 13 wherein the change in the fluorescence parameter is detected in real-time.

23. The method of claim 13 wherein the change in the fluorescence parameter is detected at a selected end-point in the amplification reaction.

24. The method of claim 13 wherein the donor fluorophore and the acceptor dye are separated by about 8-20 nucleotides in the first primer.

25. The method of claim 24 wherein the donor fluorophore and the acceptor dye are separated by about 10-16 nucleotides in the first primer.

26. The method of claim 13 wherein the donor fluorophore is fluorescein and the acceptor dye is Rhodamine X.

27. The method of claim 26 wherein the donor fluorophore and the acceptor dye are separated by 11 nucleotides in the first primer.

28. The method of claim 13 wherein the donor fluorophore is fluorescein and the acceptor dye is DABCYL.

29. The method of claim 28 wherein the donor fluorophore and the acceptor dye are separated by 11 nucleotides in the first primer.

30. The method of claim 13 wherein the donor fluorophore is Rhodamine X and the acceptor dye is Cy5.

31. The method of claim 30 wherein the donor fluorophore and the acceptor dye are separated by 11 nucleotides in the first primer.

32. The method of claim 13 wherein a change in donor fluorophore or acceptor dye fluorescence intensity is detected as an indication of amplification of the target sequence.

33. The method of claim 32 wherein the change in the fluorescence intensity is detected i) as an increase in a ratio of donor fluorophore fluorescence after cleaving or nicking to donor fluorophore fluorescence in the first primer, or ii) as a decrease in a ratio of acceptor dye fluorescence after cleaving or nicking to acceptor dye fluorescence in the first primer.

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

a) hybridizing to the target sequence a first primer comprising a donor fluorophore and an acceptor dye, the donor fluorophore and the acceptor dye linked to the first primer such that fluorescence of the donor fluorophore is quenched;

b) extending the hybridized first primer on the target sequence with a polymerase to produce a first primer extension product and separating the first primer extension product from the target sequence;

c) rendering the separated first primer extension product double-stranded by hybridization and extension of a second primer, thereby reducing donor fluorophore quenching 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.

35. The method of claim 34 wherein the donor fluorophore and the acceptor dye are an homologous dye pair.

36. The method of claim 35 wherein a change in donor fluorophore or acceptor dye fluorescence intensity is detected as an indication of amplification of the target sequence.

37. The method of claim 36 wherein the change in fluorescence intensity is detected i) as an increase in a ratio of donor fluorophore fluorescence in the double-stranded first primer extension product to donor fluorophore fluorescence in the first primer, or ii) as a decrease in a ratio of acceptor dye fluorescence in the double-stranded first primer extension product to acceptor dye fluorescence in the first primer.

38. The method of claim 35 wherein the donor fluorophore and the acceptor dye are selected from the group consisting of Cy5, Rhodamine X and fluorescein.

39. The method of claim 38 wherein the donor fluorophore and the acceptor dye are Rhodamine X.

40. The method of claim 34 wherein the donor fluorophore and the acceptor dye are a heterologous dye pair.

41. The method of claim 40 wherein a change in donor fluorophore or acceptor dye fluorescence intensity is detected as an indication of amplification of the target sequence.

42. The method of claim 41 wherein the change in fluorescence intensity is detected i) as an increase in a ratio of donor fluorophore fluorescence in the double-stranded first primer extension product to donor fluorophore fluorescence in the first primer, or ii) as a decrease in a ratio of acceptor dye fluorescence in the double-stranded first primer extension product to acceptor dye fluorescence in the first primer.

43. The method of claim 40 wherein the donor fluorophore is fluorescein and the acceptor dye is Rhodamine X or DABCYL.

44. The method of claim 34 wherein the first primer is a signal primer.

45. The method of claim 44 wherein the second primer is an amplification primer.

46. The method of claim 34 wherein the first and second primers are amplification primers.

47. A single-stranded oligonucleotide comprising:

(a) a target binding sequence;

(b) a restriction endonuclease recognition site 5'"'"' to the target binding sequence, and;

(c) a first dye and a second dye linked to the oligonucleotide at positions flanking the restriction endonuclease recognition site such that fluorescence of the first or the second dye is quenched.

48. The oligonucleotide of claim 47 wherein the first and second dyes are about 8-20 nucleotides apart in the oligonucleotide.

49. The oligonucleotide of claim 48 wherein the first and second dyes are about 10-16 nucleotides apart in the oligonucleotide.

50. The oligonucleotide of claim 49 wherein the first and second dyes are 11 nucleotides apart in the oligonucleotide.

51. The oligonucleotide of claim 47 wherein the first dye is fluorescein and the second dye is Rhodamine X or DABCYL.