Key probe compositions and methods for polynucleotide detection

• US 20060292592A1
• Filed: 02/09/2006
• Published: 12/28/2006
• Est. Priority Date: 02/09/2005
• Status: Active Grant

• Current Assignee: Stratagene California
• Original Assignee: Stratagene California
• Inventors: Sorge, Joseph A., Firmin, Andrew, Happe, Scott B.
• Granted Patent: US 7,504,218 B2
• Time in Patent Office: Days
• 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 2525/301 Hairpin oligonucleotides

Abstract

The invention relates to compositions and methods for detection of nucleic acid sequences. The invention further relates to kit format of said compositions for detection of nucleic acid sequences. Oligonucleotide probes of the invention comprise a target binding sequence and a sequence at least partially complementary thereto, joined by an optional linker to form a hairpin structure in the absence of the target nucleic acid sequence. The probes of the invention comprise a pair of moieties that produce a detectable signal when the probe hybridizes to the target sequence.

Claims

1. A probe capable of binding to a target nucleic acid sequence, said probe comprising (a) a first sequence which is at least partially complementary to said target sequence;

(b) a second sequence which is at least partially complementary to the first sequence;

(c) a first moiety operatively coupled to the first sequence; and

(d) a second label operatively coupled to the second sequence;

wherein either the 3′

end of the first sequence is linked to the 5′

end of the second sequence or the 5′

end of the first sequence is linked to the 3′

end of the second sequence;

wherein the first sequence and the second sequence are capable of hybridizing to each other when the probe is not hybridized to the target sequence; and

wherein hybridization of the probe to the target sequence causes either the first moiety or the second moiety to produce a detectable signal.

2. The probe of claim 1, further comprising a linker sequence between the first sequence and the second sequence, which linker sequence does not hybridize to either the target sequence, the first sequence, or the second sequence.

3. The probe of claim 1, further comprising a linker sequence between the first sequence and the second sequence, wherein no more than 5 contiguous nucleotides of said linker sequence are complementary to said target sequence.

4. The probe of claim 1, wherein the 3′

end of the first sequence is directly linked via a phosphodiester bond to the 5′

end of the second sequence, or wherein the 5′

end of the first sequence is directly linked via a phosphodiester bond to the 3′

end of the second sequence.

5. The probe of claim 1, wherein the first and second moieties are attached at opposite ends of the probe.

6. The probe of claim 1, wherein the first sequence is at least 5 nucleotides in length.

7. The probe of claim 6, wherein the first sequence is from about 10 to about 15 nucleotides in length.

8. The probe of claim 7, which comprises at least one modified nucleotide base that increases the Tm of the probe hybridized to the target sequence.

9. The probe of claim 6, wherein the first sequence is from about 15 to about 60 nucleotides in length.

10. The probe of claim 6, wherein the first sequence is from about 15 to about 30 nucleotides in length.

11. The probe of claim 1, wherein the second sequence is at least 5 nucleotides in length.

12. The probe of claim 1, wherein the second sequence is the same length as the first sequence.

13. The probe of claim 2, wherein the linker sequence is from about 1 to about 100 nucleotides in length.

14. The probe of claim 13, wherein the linker sequence is from about 4 to about 10 nucleotides in length.

15. The probe of claim 14, wherein the linker sequence is 5 nucleotides in length.

16. The probe of claim 1, wherein the first sequence is fully complementary over its entire length to the target sequence.

17. The probe of claim 1, wherein the first sequence is fully complementary over its entire length to the second sequence.

18. The probe of claim 1, wherein the second sequence is identical to the target sequence.

19. The probe of claim 1, comprising at least 1 mismatch between the first sequence and the second sequence.

20. The probe of claim 19, comprising from about 3 to about 6 mismatches.

21. The probe of claim 19, comprising from about 2 to about 8 mismatches.

22. The probe of claim 19, wherein the number of mismatches corresponds to from about 10% to about 30% of the length of the first sequence.

23. The probe of claim 1, wherein the Tm of the stem structure is less than the Tm of the probe hybridized to the target sequence.

24. The probe of claim 1, wherein the affinity of the first sequence for the target sequence is greater that the affinity of the first sequence for the second sequence.

25. The probe of claim 1, wherein the either the first or second moiety is a fluorophore and the other moiety is a quencher.

26. The probe of claim 1, wherein the detectable signal is an increase in fluorescence intensity.

27. The probe of claim 1 which produces a 5′

flap when bound to the target sequence.

28. The probe of claim 27, wherein a detectable signal is produced upon invasive cleavage with a flap endonuclease.

29. The probe of claim 27, wherein a detectable signal is produced upon non-invasive cleavage with a flap endonuclease.

30. The probe of claim 1 which produces a 3′

flap when bound to the target sequence.

31. A method of detecting a target nucleic acid sequence, comprising the steps of (1) incubating the target sequence with a probe capable of binding to a target nucleic acid sequence, said probe comprising (a) a first sequence which is at least partially complementary to a target sequence;

(b) a second sequence which is at least partially complementary to the first sequence;

(c) a first moiety operatively coupled to the first sequence; and

(d) a second moiety operatively coupled to the second sequence;

wherein either the 3′

end of the first sequence is linked to the 5′

end of the second sequence or the 5′

end of the first sequence is linked to the 3′

end of the second sequence;

wherein the first sequence and the second sequence are capable of hybridizing to each other when the probe is not hybridized to the target sequence; and

wherein hybridization of the probe to the target sequence causes either the first moiety or the second moiety to produce a detectable signal; and

(2) detecting a signal from the first or second moiety.

32. The method of claim 31 wherein the 5′

end of the first sequence is linked to the 3′

end of the second sequence.

33. The method of claim 31 wherein the step of incubating is performed in the presence of a nuclease.

34. The method of claim 33 wherein the nuclease is a flap endonuclease.

35. The method of claim 34 wherein a detectable signal is produced if the flap endonuclease performs an invasive cleavage.

36. The method of claim 34 wherein a detectable signal is produced if the flap endonuclease performs a non-invasive cleavage.

37. A method of detecting a target nucleic acid sequence, comprising the steps of (1) amplifying the target sequence using a polymerase chain reaction in the presence of a probe capable of binding to the target sequence, said probe comprising (a) a first sequence which is at least partially complementary to the target sequence and which consists of a stem portion and a loop portion;

(b) a second sequence which is shorter than the first sequence and at least partially complementary to the stem portion of the first sequence; and

(c) first and second moieties;

wherein the 3′

end of the first sequence is linked to the 5′

end of the second sequence;

wherein the first sequence and the second sequence are capable of hybridizing to each other when the probe is not hybridized to the target sequence; and

wherein hybridization of the probe to the target sequence causes the first or second moiety to produce a detectable signal; and

(2) detecting a signal from the first or second moiety.

38. The method of claim 37 wherein the 5′

end of the first sequence is linked to the 3′

end of the second sequence.

39. The method of claim 37 wherein the step of incubating is performed in the presence of a nuclease.

40. The method of claim 37 wherein the nuclease is a flap endonuclease.

41. The method of claim 40 wherein a detectable signal is produced if the flap endonuclease produces an invasive cleavage.

42. The method of claim 40 wherein a detectable signal is produced if the flap endonuclease produces a non-invasive cleavage.