Dual labeled fluorescent probes

• US 20060088856A1
• Filed: 08/12/2005
• Published: 04/27/2006
• Est. Priority Date: 08/13/2004
• Status: Active Grant

• Current Assignee: Hologic Incorporated
• Original Assignee: Stratagene California
• Inventors: Mueller, Reinhold, Sorge, Joseph A., Padmabandu, Gothami
• Granted Patent: US 7,361,469 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

The present invention relates to probes useful for the detection and measurement of target nucleic acids, as well as compositions and kits containing such probes. The probes of the present invention include an interactive pair of labels, as well as a hairpin sequence that does not hybridize to a target nucleic acid. According to the present invention, the probe generates a detectable signal indicative of a presence of a target nucleic acid. The detectable signal emitted by one of the pair of labels is substantially constant upon binding of the probe to target nucleic acid, and the detectable signal increases by at least 2 fold upon cleavage of the probe between the pair of labels.

Claims

1. An oligonucleotide probe having a 5′

and 3′

end, comprising;

(a) a hairpin sequence that does not hybridize to a target nucleic acid and consisting of a stem and a loop;

(b) a target binding sequence that forms a hybrid with a target nucleic acid; and

,

(c) an interactive pair of labels, consisting of a first and second member;

 

wherein said hairpin sequence and said target binding sequence are covalently linked, wherein said first member of said pair is attached between said hairpin sequence and said target binding sequence and wherein said second member of said pair is attached to said target binding sequence at the terminus distal to said hairpin sequence, wherein a detectable signal emitted by one of said pair of labels is substantially constant upon binding of said probe to target nucleic acid, and wherein said detectable signal increases by at least 2 fold upon cleavage of said probe between said pair.

2. The probe of claim 1, further comprising a linker sequence, located between said hairpin sequence and said target binding sequence.

3. The probe of claim 2, wherein said linker sequence comprises between 1 and 15 nucleotides.

4. The probe of claim 2, wherein said linker sequence comprises between 1 and 10 nucleotides.

5. The probe of claim 2, wherein said linker sequence comprises between 1 and 5 nucleotides.

6. The probe of claim 1, wherein said pair comprises a fluorophore and a quencher.

7. The probe of claim 6, wherein of one of said fluorophore or said quencher is attached to said 3′

nucleotide such that said probe does not serve as a template for synthesis.

8. The probe of claim 7, wherein said fluorophore is attached to said 3′

nucleotide.

9. The probe of claim 1, wherein said pair is separated by between 5 and 60 nucleotides.

10. The probe of claim 1, wherein said pair is separated by between 10 and 30 nucleotides.

11. The probe of claim 6, wherein said fluorophore is selected from the group consisting of FAM, R110, TAMRA, R6G, CAL Fluor Red 610, CAL Fluor Gold 540, and CAL Fluor Orange 560.

12. The probe of claim 6, wherein said quencher is selected from the group consisting of DABCYL, BHQ-1, BHQ-2, and BHQ-3.

13. The probe of claim 6, wherein said fluorescence increases upon cleavage of said probe between said fluorophore and quencher by at least 3 fold.

14. The probe of claim 6, wherein said fluorescence increases upon cleavage of said probe between said fluorophore and quencher by at least 4 fold.

15. The probe of claim 1, wherein said stem of said hairpin sequence is between 2 to 20 base pairs.

16. The probe of claim 1, wherein said stem of said hairpin sequence is between 3 to 10 base pairs.

17. The probe of claim 1, wherein said stem of said hairpin sequence is between 4 to 8 base pairs.

18. The probe of claim 1, wherein said loop of said hairpin sequence is between 2 and 30 bases.

19. The probe of claim 1, wherein said loop of said hairpin sequence is between 3 and 20 bases.

20. The probe of claim 1, wherein said loop of said hairpin sequence is between 3 and 10 bases.

21. The probe of claim 6, wherein one of said fluorophore or quencher is attached at the 3′

OH moiety of said target binding sequence.

22. The probe of claim 6, wherein one of said fluorophore or quencher is attached to the linker sequence.

23. The probe of claim 6, wherein one of said fluorophore or quencher is attached to the target binding sequence.

24. The probe of claim 6, wherein said fluorophore or quencher is directly attached.

25. The probe of claim 6, wherein said fluorophore or quencher is indirectly attached via a spacer.

26. The probe of claim 6, further comprising a solid support.

27. The probe of claim 26, wherein said solid support is attached to the 5′

end of said hairpin sequence.

30. A method of detecting a target nucleic acid, by incubating the probe of claim 1 under conditions which permit hybridization of said probe to target in a sample comprising a target nucleic acid with a nucleic acid polymerase and a FEN nuclease and cleaving said probe between said pair of labels with a FEN nuclease to generate a signal, wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.

31. The method of claim 30 wherein said nucleic acid polymerase substantially lacks 5′

to 3′

exonuclease activity.

32. The method of claim 30 wherein the nucleic acid polymerase is a DNA polymerase.

33. The method of claim 30 wherein the nucleic acid polymerase is thermostable.

34. The method of claim 30 wherein the FEN nuclease is a flap-specific nuclease.

35. The method of claim 30 wherein the FEN nuclease is thermostable.

36. A composition comprising the probe of claim 1 and a primer.

37. The composition of claim 36, further comprising a nucleic acid polymerase.

38. The composition of claim 31, wherein said nucleic acid polymerase is a DNA polymerase.

39. The composition of claim 36, wherein said nucleic acid polymerase substantially lacks 5′

to 3′

exonuclease activity.

40. The composition of claim 36, further comprising FEN nuclease.

41. A composition comprising the probe of claim 1, and a nucleic acid polymerase.

42. The composition of claim 41, wherein said nucleic acid polymerase substantially lacks 5′

to 3′

exonuclease activity.

43. The composition of claim 41, further comprising a FEN nuclease.

44. The composition of claim 41, further comprising a primer.

45. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising the probe of claim 1, a nucleic acid polymerase substantially lacking 5′

to 3′

exonuclease activity, and packaging material therefor.

46. The kit of claim 45, further comprising a suitable buffer.

47. The kit of claim 45, further comprising a FEN nuclease.

48. The kit of claim 45, further comprising a primer.

49. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising the probe of claim 1, a FEN nuclease, and packaging material therefor.

50. The kit of claim 49, further comprising a suitable buffer.

51. The kit of claim 49, further comprising a nucleic acid polymerase substantially lacking 5′

to 3′

exonuclease activity.

52. The kit of claim 49, further comprising a primer.

53. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising the probe of claim 1, a primer, and packaging material therefor.

54. The kit of claim 53, further comprising a suitable buffer.

55. The kit of claim 53, further comprising a nucleic acid polymerase substantially lacking 5′

to 3′

exonuclease activity.

56. The kit of claim 53, further comprising a FEN nuclease.

28. An oligonucleotide probe having a 5′

and 3′

end, comprising;

(a) a hairpin sequence that does not hybridize to a target nucleic acid and consisting of a stem and a loop;

(b) a target binding sequence that forms a hybrid with a target nucleic acid; and

,

(c) an interactive pair of labels, consisting of a first and second member;

 

wherein said hairpin sequence and said target binding sequence are covalently linked such that said hairpin sequence is positioned 5′

of said target binding sequence, wherein said first member of said pair is attached to the 3′

terminus of said target binding sequence and wherein said second member of said pair is attached to said oligonucleotide probe between said 3′

terminus and said hairpin sequence, and wherein a detectable signal emitted by one of said pair of labels is substantially constant upon binding of said probe to target nucleic acid, and wherein said signal increases by at least 2 fold upon cleavage of said probe between said pair.

29. An oligonucleotide probe having a 5′

and 3′

end, comprising;

(a) a hairpin sequence that does not hybridize to a target nucleic acid and consisting of a stem and a loop;

(b) a target binding sequence that forms a hybrid with a target nucleic acid; and

,

(c) an interactive pair of labels, consisting of a first and second member;

 

wherein said hairpin sequence and said target binding sequence are covalently linked such that said hairpin sequence is positioned 3′

of said target binding sequence;

wherein said first member of said pair is attached to the 5′

terminus of said target binding sequence and wherein said second member of said pair is attached to said oligonucleotide probe between the 3′

terminus of said target binding sequence and said hairpin sequence;

wherein a detectable signal emitted by one of said pair of labels is substantially constant upon binding of said probe to target nucleic acid; and

 

wherein said detectable signal increases by at least 2 fold upon cleavage of said probe between said pair.