Methods and probes for detection and/or quantification of nucleic acid sequences

• US 6,815,164 B2
• Filed: 10/09/2001
• Issued: 11/09/2004
• Est. Priority Date: 10/06/2000
• Status: Expired due to Term

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): STRZELECKA, TERESA E
• Application Number:
  US09/974,756
• Publication Number:
  US 20020115088A1
• Time in Patent Office: 1,127 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2537/1376 Displacement by an enzyme
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/163 blocking probe
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

The present invention discloses nucleic acid detector probes for specific detection and/or quantification of target nucleic acid sequences and detection and/or quantification methods using these probes. In the absence of target nucleic acid sequence, a first oligonucleotide and a third oligonucleotide are bound to each other in a conformation which brings two member of an interacting moiety pair (labels) into close spatial proximity. Cooperative binding of the first oligonucleotide and a second oligonucleotide to a target nucleic acid sequence causes displacement of the third oligonucleotide from the first oligonucleotide probe resulting in separation of the two members of the interacting moiety pair (labels). The spatial separation of the moieties (labels) is detectable, and indicates the presence and/or amount of the target nucleic acid sequence. The method is useful for detection and/or quantification of a specific nucleic acid sequence as well as the detection of sequence alteration in the target nucleic acid sequence.

Claims

1. A method of determining whether a target nucleic acid sequence is present in a sample, said method comprising:

contacting said sample with a first probe, a second probe, and a third probe, under conditions allowing hybridization of the first and second probes to the target nucleic acid sequence, if present, and hybridization of the first probe to the third probe, in the absence of the target nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to the target nucleic acid sequence, wherein;

(i) the first probe comprises

(a) a polynucleotide comprising a 3′

region which hybridizes to a first region of the target nucleic acid sequence, if present, and

(b) a first member of an interacting label pair;

(ii) the second probe comprises

(a) a polynucleotide comprising a 5′

region which hybridizes to a second region of the target nucleic acid sequence, if present, and

(b) a 3′

region which hybridizes to a sequence in the 5′

region of the first probe, if the target nucleic acid sequence is present; and

(iii) the third probe comprises

(a) a polynucleotide which hybridizes to a sequence in the 5′

region of the first probe, and

(b) a second member of an interacting label pair;

wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact;

and wherein, in the presence of the target nucleic acid sequence, said 3′

region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 5′

region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 3′

region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.

3. The method of claim 1 or 2, wherein said first and second regions of the target nucleic acid sequence are contiguous.

4. The method of claim 1 or 2, wherein said first and second regions of the target nucleic acid sequence are 1 nucleotide apart.

5. The method of claim 1 or 2, wherein said first and second regions of the target nucleic acid sequence are 3 or fewer nucleotides apart.

6. The method of claim 1 or 2, wherein said first and second regions of the target nucleic acid sequence are 7 or fewer nucleotides apart.

7. The method of claim 1 or 2, wherein the interacting label pair comprises donor and acceptor moieties.

8. The method of claim 1 or 2, wherein the interacting label pair comprises an enzyme.

9. The method of claim 8, wherein the interacting label pair comprises an enzyme and an inhibitor of said enzyme.

10. The method of claim 1 or 2, wherein the interacting label pair is capable of energy transfer.

11. The method of claim 1 or 2, wherein the interacting label pair comprises a fluorophore and a quencher.

12. The method of claim 1 or 2, wherein the method further comprises measuring the magnitude of the signal generated, whereby said magnitude indicates the quantity of the target nucleic acid sequence.

13. The method of claim 1, wherein the method further comprises extension of the 3′

end of the second probe along a sequence in the 5′

region of the first probe using a nucleotide polymerase.

14. The method of claim 1 or 2, wherein the target nucleic acid sequence is attached to an analyte.

15. The method of claim 1 or 2, wherein the target nucleic acid sequence is attached to a solid support.

16. The method of claim 1 or 2, wherein the target nucleic acid sequence comprises DNA.

17. The method of claim 1 or 2, wherein the target nucleic acid sequence comprises RNA.

18. The method of claim 1 or 2, wherein the target nucleic acid sequence comprises DNA and RNA.

19. The method of claim 1 or 2, wherein the target nucleic acid sequence comprises PNA.

20. The method of claim 1 or 2, wherein at least one of the probes comprises DNA.

21. The method of claim 1 or 2, wherein at least one of the probes comprises RNA.

22. The method of claim 1 or 2, wherein at least one of the probes comprises DNA and RNA.

23. The method of claim 1 or 2, wherein at least one of the probes comprises PNA.

24. The method of claim 1 or 2, wherein the region of the second probe which is hybridizable to a sequence of the first probe comprises a modified nucleotide that causes enhanced affinity to the sequence in the region of the first probe relative to an unmodified nucleotide.

25. The method of claim 1 or 2, wherein said detectable signal is of a greater magnitude than a detectable signal associated with the interacting label pair when the third probe is hybridized to the first probe.

26. The method of claim 1 or 2, wherein said detectable signal is of a lesser magnitude than a detectable signal associated with the interacting label pair when the third probe is hybridized to the first probe.

27. The method of claim 1 or 2, wherein the method further comprises amplifying the target nucleic acid sequence.

28. The method of claim 1 or 2, wherein the first probe or the second probe is allele-specific.

29. The method of claim 1 or 2, wherein the first probe and the second probe are allele-specific.

2. A method of determining whether a target nucleic acid sequence is present in a sample, said method comprising:

contacting said sample with a first probe, a second probe, and a third probe, under conditions allowing hybridization of the first and second probes to the target nucleic acid sequence, if present, and hybridization of the first probe to the third probe, in the absence of the target nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to the target nucleic acid sequence, wherein;

(i) the first probe comprises

(a) a polynucleotide comprising a 5′

region which hybridizes to a first region of the target nucleic acid sequence, if present, and

(b) a first member of an interacting label pair;

(ii) the second probe comprises

(a) a polynucleotide comprising a 3′

region which hybridizes to a second region of the target nucleic acid sequence, if present, and

(b) a 5′

region which hybridizes to a sequence in the 3′

region of the first probe, if the target nucleic acid sequence is present; and

(iii) the third probe comprises

(a) a polynucleotide which hybridizes to a sequence in the 3′

region of the first probe, and

(b) a second member of an interacting label pair;

wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact;

and wherein, in the presence of the target nucleic acid sequence, said 5′

region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 3′

region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 5′

region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.

30. A method of determining whether a target nucleic acid sequence is present in a sample, said method comprising:

contacting said sample with a first probe, a second probe, a third probe, and a nucleotide polymerase, under conditions allowing hybridization of the first and second probes to the target nucleic acid sequence, if present, and hybridization of the first probe to the third probe, in the absence of the target nucleic acid sequence, and allowing nucleic acid polymerization, wherein;

(i) the first probe comprises

(a) a polynucleotide comprising a 3′

region which hybridizes to a first region of the target nucleic acid sequence, if present,

(b) a first member of an interacting label pair; and

(ii) the second probe comprises a polynucleotide comprising a 5′

region which hybridizes to a second region of the target nucleic acid sequence, if present; and

(iii) the third probe comprises

(a) a polynucleotide sequence which hybridizes to a sequence in the 5′

region of the first probe, and

(b) a second member of an interacting label pair;

wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact;

and wherein, in the presence of the target nucleic acid sequence, said 3′

region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 5′

region of said second probe hybridizes to said second region of the target nucleic acid sequence;

and wherein the 3′

end of said second probe is extended by said nucleotide polymerase along the first probe by template switching, causing dissociation of the first and second members of the interacting label pair;

whereby generation of detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.

31. A method of determining whether a target nucleic acid sequence is present in a sample, said method comprising:

contacting said sample with a first probe and a second probe, under conditions allowing hybridization of the first and second probes to the target nucleic acid sequence, if present, and hybridization of a first nucleotide sequence in the 5′

region of the first probe with a second nucleotide sequence in the 5′

region of the first probe, in the absence of the target nucleic acid sequence, and hybridization of at the second probe the first probe when the second probe is hybridized to the target nucleic acid sequence, wherein;

(i) the first probe comprises a polynucleotide comprising

(a) a 3′

region which hybridizes to a first region of the target nucleic acid sequence, if present, and

(b) a 5′

region comprising said first and second nucleotide sequences which hybridize to each other in the absence of the target nucleic acid sequence, and two members of an interacting label pair;

(ii) the second probe comprises a polynucleotide comprising

(a) a 5′

region which hybridizes to a second region of the target nucleic acid sequence, if present and

(b) a 3′

region which hybridizes to a sequence in the 5′

region of the first probe, if the target polynucleotide is present;

wherein, when said first and second nucleotide sequences in the 5′

region of the first probe hybridize, said first and second members of the interacting label pair are brought into proximity and interact; and

wherein, in the presence of the target nucleic acid sequence, said first probe and said second probe hybridize to the target nucleic acid sequence and said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

whereby generation of detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.

32. A method of determining whether a target nucleic acid sequence is present in a sample, said method comprising:

contacting said sample with a first probe and a second probe, under conditions allowing hybridization of the first and second probes to the target nucleic acid sequence, if present, and hybridization of a first nucleotide sequence in the 3′

region of the first probe with a second nucleotide sequence in the 3′

region of the first probe, in the absence of the target nucleic acid sequence, and hybridization of at the second probe the first probe when the second probe is hybridized to the target nucleic acid sequence, wherein;

(i) the first probe comprises a polynucleotide comprising

(a) a 5′

region which is hybridizes to a first region of the target nucleic acid sequence, if present, and

(b) a 3′

region comprising said first and second nucleotide sequences which hybridize to each other in the absence of the target nucleic acid sequence, and two members of an interacting label pair;

(ii) the second probe comprises a polynucleotide comprising

(a) a 3′

region which hybridizes to a second region of the target nucleic acid sequence, if present and

(b) a 5′

region which hybridizes to a sequence in the 3′

region of the first probe, if the target polynucleotide is present;

wherein, when said first and second nucleotide sequences in the 3′

region of the first probe hybridize, said first and second members of the interacting label pair are brought into proximity and interact; and

wherein, in the presence of the target nucleic acid sequence, said first probe and said second probe hybridize to the target nucleic acid sequence and said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

whereby generation of detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.

33. A method of determining whether a target nucleic acid sequence contains a sequence alteration relative to a reference nucleic acid sequence, said method comprising:

(a) contacting said target nucleic acid sequence with a first probe, a second probe, and a third probe, and

(b) contacting said reference nucleic acid sequence with a first probe, a second probe, and a third probe,

wherein said contact of the target nucleic acid sequence with the first, second, and third probes, and said contact of the reference nucleic acid sequence with the first, second, and third probes, occur under conditions allowing hybridization of the first and second probes to the reference nucleic acid sequence, and hybridization of the first probe to the third probe, in the absence of the reference nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to the reference nucleic acid sequence, wherein;

(i) the first probe comprises

(a) a polynucleotide comprising a 3′

region which hybridizes to a first region of the reference nucleic acid sequence, if present, and

(b) a first member of an interacting label pair;

(ii) the second probe comprises

(a) a polynucleotide comprising a 5′

region which hybridizes to a second region of the reference nucleic acid sequence, if present, and

(b) a 3′

region which hybridizes to a sequence in the 5′

region of the first probe, if the reference nucleic acid sequence is present; and

(iii) the third probe comprises

(a) a polynucleotide which hybridizes to a sequence in the 5′

region of the first probe, and

(b) a second member of an interacting label pair;

wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact;

and wherein, in the presence of the reference nucleic acid sequence, said 3′

region of said first probe hybridizes to said first region of the reference nucleic acid sequence and said 5′

region of said second probe hybridizes to said second region of the reference nucleic acid sequence and said 3′

region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

and comparing detectable signal generated by contacting said first, second, and third probes with the reference nucleic acid sequence with detectable signal generated by contacting said first, second, and third probes with target nucleic acid sequence;

whereby reduced signal generation by the target nucleic acid sequence as compared to the reference nucleic acid sequence indicates the presence of an altered sequence in the target nucleic acid sequence relative to the reference nucleic acid sequence.

34. A method of determining whether a target nucleic acid sequence contains a sequence alteration relative to a reference nucleic acid sequence, said method comprising:

(a) contacting said target nucleic acid sequence with a first probe, a second probe, and a third probe, and

(b) contacting said reference nucleic acid sequence with a first probe, a second probe, and a third probe,

wherein said contact of the target nucleic acid sequence with the first, second, and third probes, and said contact of the reference nucleic acid sequence with the first, second, and third probes, occur under conditions allowing hybridization of the first and second probes to the reference nucleic acid sequence, and hybridization of the first probe to the third probe, in the absence of the reference nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to the reference nucleic acid sequence, wherein;

(i) the first probe comprises

(a) a polynucleotide comprising a 5′

region which hybridizes to a first region of the reference nucleic acid sequence, if present, and

(b) a first member of an interacting label pair;

(ii) the second probe comprises

(a) a polynucleotide comprising a 3′

region which hybridizes to a second region of the reference nucleic acid sequence, if present, and

(b) a 5′

region which hybridizes to a sequence in the 3′

region of the first probe, if the reference nucleic acid sequence is present; and

(iii) the third probe comprises

(a) a polynucleotide which hybridizes to a sequence in the 5′

region of the first probe, and

(b) a second member of an interacting label pair;

wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact;

and wherein, in the presence of the reference nucleic acid sequence, said 5′

region of said first probe hybridizes to said first region of the reference nucleic acid sequence and said 3′

region of said second probe hybridizes to said second region of the reference nucleic acid sequence and said 5′

region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

and comparing detectable signal generated by contacting said first, second, and third probes with the reference nucleic acid sequence with detectable signal generated by contacting said first, second, and third probes with target nucleic acid sequence;

whereby reduced signal generation by the target nucleic acid sequence as compared to the reference nucleic acid sequence indicates the presence of an altered sequence in the target nucleic acid sequence relative to the reference nucleic acid sequence.

35. A method of determining whether one or more of a plurality of target nucleic acid sequences is present in a sample, said method comprising:

contacting said sample with a plurality of probe sets, each set comprising a first probe, a second probe, and a third probe, under conditions allowing hybridization of the first and second probes to a target nucleic acid sequence, if present, and hybridization of the first probe to the third probe, in the absence of said target nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to said target nucleic acid sequence, wherein;

(i) the first probe comprises

(a) a polynucleotide comprising a 3′

region which hybridizes to a first region of said target nucleic acid sequence, if present, and

(b) a first member of an interacting label pair;

(ii) the second probe comprises

(a) a polynucleotide comprising a 5′

region which hybridizes to a second region of said target nucleic acid sequence, if present, and

(b) a 3′

region which hybridizes to a sequence in the 5′

region of the first probe, if said target nucleic acid sequence is present;

(iii) the third probe comprises

(a) a polynucleotide which hybridizes to a sequence in the 5′

region of the first probe, and

(b) a second member of an interacting label pair;

wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact;

and wherein, in the presence of said target nucleic acid sequence, said 3′

region of said first probe hybridizes to said first region of said target nucleic acid sequence and said 5′

region of said second probe hybridizes to said second region of said target nucleic acid sequence and said 3′

region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of said target nucleic acid sequence; and

wherein each probe set comprises an interacting label pair which generates a detectable signal which is different from the signals of the interacting label pairs of every other probe set, and generation of two or more signals indicates presence of a plurality of target nucleic acid sequences.

36. A method of determining whether one or more of a plurality of target nucleic acid sequences is present in a sample, said method comprising:

contacting said sample with a plurality of probe sets, each set comprising a first probe, a second probe, and a third probe, under conditions allowing hybridization of the first and second probes to a target nucleic acid sequence, if present, and hybridization of the first probe to the third probe, in the absence of said target nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to said target nucleic acid sequence, wherein;

(i) the first probe comprises

(a) a polynucleotide comprising a 5′

region which hybridizes to a first region of said target nucleic acid sequence, if present, and

(b) a first member of an interacting label pair;

(ii) the second probe comprises

(a) a polynucleotide comprising a 3′

region which hybridizes to a second region of said target nucleic acid sequence, if present, and

(b) a 5′

region which hybridizes to a sequence in the 3′

region of the first probe, if said target nucleic acid sequence is present;

(iii) the third probe comprises

(a) a polynucleotide which hybridizes to a sequence in the 3′

region of the first probe, and

(b) a second member of an interacting label pair;

wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact;

and wherein, in the presence of said target nucleic acid sequence, said 5′

region of said first probe hybridizes to said first region of said target nucleic acid sequence and said 3′

region of said second probe hybridizes to said second region of said target nucleic acid sequence and said 5′

region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of said target nucleic acid sequence; and

wherein each probe set comprises an interacting label pair which generates a detectable signal which is different from the signals of the interacting label pairs of every other probe set, and generation of two or more signals indicates presence of a plurality of target nucleic acid sequences.

39. The composition of claim 36 or 37, wherein the interacting moiety pair comprises a fluorophore and a quencher.

40. The composition of claim 36, further comprising a nucleotide polymerase.

41. The composition of claim 36 or 37, further comprising the target nucleic acid sequence.

42. The composition of claim 36 or 37, further comprising a reference nucleic acid sequence to which the target nucleic acid sequence is to be compared.

37. A composition comprising a first probe, a second probe and a third probe, wherein:

(i) the first probe comprises

(a) a polynucleotide comprising a 3′

region which hybridizes to a first region of a target nucleic acid sequence, if present, and

(b) a first member of an interacting label pair;

(ii) the second probe comprises

(a) a polynucleotide comprising a 5′

region which hybridizes to a second region of the target nucleic acid sequence, if present, and

(b) a 3′

region which hybridizes to a sequence in the 5′

region of the first probe, if the target nucleic acid sequence is present;

(iii) the third probe comprises

(a) a polynucleotide which hybridizes to a sequence in the 5′

region of the first probe, and

(b) a second member of an interacting label pair;

wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact;

and wherein, in the presence of the target nucleic acid sequence, said 3′

region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 5′

region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 3′

region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.

38. A composition comprising a first probe, a second probe and a third probe, wherein:

(i) the first probe comprises

(a) a polynucleotide comprising a 5′

region which hybridizes to a first region of a target nucleic acid sequence, if present, and

(b) a first member of an interacting label pair;

(ii) the second probe comprises

(a) a polynucleotide comprising a 3′

region which hybridizes to a second region of the target nucleic acid sequence, if present, and

(b) a 5′

region which hybridizes to a sequence in the 3′

region of the first probe, if the target nucleic acid sequence is present;

(iii) the third probe comprises

(a) a polynucleotide which hybridizes to a sequence in the 3′

region of the first probe, and

(b) a second member of an interacting label pair;

wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact;

and wherein, in the presence of the target nucleic acid sequence, said 5′

region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 3′

region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 5′

region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.

43. A kit for determining whether a target nucleic acid is present in a sample or quantifying a target nucleic acid sequence, comprising a first probe, a second probe and a third probe, wherein:

(i) the first probe comprises

(a) a polynucleotide comprising a 3′

region which hybridizes to a first region of a target nucleic acid sequence, if present, and

(b) a first member of an interacting label pair;

(ii) the second probe comprises

(a) a polynucleotide comprising a 5′

region which hybridizes to a second region of the target nucleic acid sequence, if present, and

(b) a 3′

region which hybridizes to a sequence in the 5′

region of the first probe, if the target nucleic acid sequence is present; and

(iii) the third probe comprises

(a) a polynucleotide which hybridizes to a sequence in the 5′

region of the first probe, and

(b) a second member of an interacting label pair;

wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact;

and wherein, in the presence of the target nucleic acid sequence, said 3′

region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 5′

region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 3′

region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.

45. The kit of claim 43 or 44, further comprising a reference nucleic acid sequence to which the target nucleic acid sequence may be compared.

46. The kit of claim 43 or 44, further comprising instructions for use of the kit to determine the presence of the target nucleic acid sequence in a sample or quantify the target nucleic acid sequence.

44. A kit for determining whether a target nucleic acid is present in a sample or quantifying a target nucleic acid sequence, comprising a first probe, a second probe and a third probe, wherein:

(i) the first probe comprises

(a) a polynucleotide comprising a 5′

region which hybridizes to a first region of a target nucleic acid sequence, if present, and

(b) a first member of an interacting label pair;

(ii) the second probe comprises

(a) a polynucleotide comprising a 3′

region which hybridizes to a second region of the target nucleic acid sequence, if present, and

(b) a 5′

region which hybridizes to a sequence in the 3′

region of the first probe, if the target nucleic acid sequence is present;

(iii) the third probe comprises

(a) a polynucleotide which hybridizes to a sequence in the 3′

region of the first probe, and

(b) a second member of an interacting label pair;

wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact;

and wherein, in the presence of the target nucleic acid sequence, said 5′

region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 3′

region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 5′

region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair;

whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.