Self-hybridizing multiple target nucleic acid probes and methods of use

• US 20050176023A1
• Filed: 07/30/2004
• Published: 08/11/2005
• Est. Priority Date: 08/01/2003
• Status: Abandoned Application

• Current Assignee: Life Technologies AS (Perspecta, Inc.)
• Original Assignee: Dynal Biotech, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Ramon, Daniel S., Wang, Lu
• Application Number:
  US10/909,278
• Publication Number:
  US 20050176023A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

The present invention provides probes for target nucleic acid sequences and methods for using the probes to detect the presence or absence of a desired target nucleic acid sequence. The present probes contain two or more probing regions. The probes also contain multiple self-complementary sequences that hybridize to one another in the absence of one or more of the target sequences. The probes can also optionally contain one or more arm sequences, spacer sequences or labels as desired.

Claims

1. A nucleic acid probe comprising:

(a) a first probe region specific for a first target sequence; and

(b) a second probe region specific for a second target sequence, wherein;

(i) the first probe region and the second probe region are non-contiguous, the first target sequence and the second target sequence are non-contiguous, or both the first probe region and the second probe region and the first target sequence and the second target sequence are non-contiguous; and

(ii) the nucleic acid probe comprises self-complementary sequences.

2. The nucleic acid probe of claim 1 wherein the first target sequence and the second target sequence are on the same target nucleic acid.

3. The nucleic acid probe of claim 2 wherein the nucleic acid probe is capable of distinguishing the target nucleic acid which comprises both the first target sequence and the second target sequence from nucleic acids that do not comprise both the first target sequence and the second target sequence.

4. The nucleic acid probe of claim 1 wherein the first target sequence and the second target sequence are on different target nucleic acids.

5. The nucleic acid probe of claim 1 wherein there are two or four self-complementary sequences.

6. The nucleic acid probe of claim 1 wherein neither the first probe region nor the second probe region comprise at least part of the self-complementary sequences.

7. The nucleic acid probe of claim 1 wherein none of the self-complementary sequences are located between the first probe region and the second probe region.

8. The nucleic acid probe of claim 1 wherein at least one of the first probe region and the second probe region comprises at least part of one of the self-complementary sequences.

9. The nucleic acid probe of claim 8 wherein only one of the first probe region and the second probe region comprises one of the self-complementary sequences.

10. The nucleic acid probe of claim 1 wherein the first probe region comprises at least part of one of the self-complementary sequences and the second probe region comprises at least part of another of the self-complementary sequences.

11. The nucleic acid probe of claim 10 wherein the self-complementary sequence of the first probe region is complementary to the self-complementary sequence of the second probe region.

12. The nucleic acid probe of claim 10 wherein the self-complementary sequence of the first probe region is not complementary to the second probe region and the self-complementary sequence of the second probe region is not complementary to the first probe region.

13. The nucleic acid probe of claim 1 further comprising:

(c) a spacer moiety that separates the first probe region and the second probe region.

14. The nucleic acid probe of claim 13 wherein the spacer moiety comprises a nucleic acid.

15. The nucleic acid probe of claim 1 further comprising

(d) one or more arm sequences that comprise one or more of the self-complementary sequences, wherein the one or more arm sequences do not comprise the probe regions.

16. The nucleic acid probe of claim 15 wherein the one or more self-complementary sequences of the one or more arm sequences are complementary with a self-complementary sequence located at least partially within one of the probe regions.

17. The nucleic acid probe of claim 15 wherein a first self-complementary sequence of a first of the one or more arm sequences is complementary with a second self-complementary sequence of a second of the one or more arm sequences.

18. The nucleic acid probe of claim 15 wherein at least one of the one or more self-complementary sequences of the one or more arm sequences is complementary to at least a portion of the spacer moiety.

19. The nucleic acid probe of claim 1 further comprising

(e) one or more detectable labels, wherein the one or more detectable labels can be the same or different.

20. The nucleic acid probe of claim 19 wherein the detectable label is covalently bound to a terminus of the nucleic acid probe.

21. The nucleic acid probe of claim 19 wherein the detectable label is covalently bound to a nucleotide in the nucleic acid probe.

22. The nucleic acid probe of claim 19 wherein the detectable label is covalently bound to a spacer moiety of the nucleic acid probe.

23. The nucleic acid probe of claim 19 wherein the detectable label comprises a linker group, wherein the linker group is covalently bound to the nucleic acid probe.

24. The nucleic acid probe of claim 23 wherein the linker group comprises an amino linker.

25. The nucleic acid probe of claim 19 wherein the detectable label is a fluorescent molecule.

26. The nucleic acid probe of claim 19 wherein the detectable label is a bead.

27. The nucleic acid probe of claim 26 wherein the bead is a latex or polystyrene bead.

28. The nucleic acid probe of claim 1 wherein the nucleic acid probe is attached to a solid support.

29. The nucleic acid probe of claim 1 wherein the first probe region and second probe region have different sequences.

30. The nucleic acid probe of claim 1 wherein the first probe region and second probe region have the same sequence.

31. The nucleic acid probe of claim 1 wherein the first target sequence and second target sequence are motifs and the target nucleic acid is an allele, further wherein that the first target sequence and second target sequence are motifs on the same allele.

32. The nucleic acid probe of claim 25 wherein the nucleic acid probe does not comprise a fluorescent quencher moiety.

33. An array of nucleic acid probes comprising one or more of a plurality of the nucleic acid probes of claim 1 located in defined, distinct locations on a solid support.

34. The array of nucleic acid probes of claim 33 wherein one or more of the plurality of nucleic acid probes is attached to a solid support.

35. The array of nucleic acid probes of claim 34 wherein one or more of the plurality of nucleic acid probes is contained within a well of the solid support.

36. The array of nucleic acid probes of claim 33 wherein the nucleic acid probes in different defined, distinct locations on the solid support have one or more different probe regions.

37. The array of nucleic acid probes of claim 33 wherein the nucleic acid probes in different defined, distinct locations on the solid support have one or more of the same probe regions.

38. The array of nucleic acid probes of claim 37 wherein one or more of the plurality of nucleic acid probes have the same probe regions.

39. The array of nucleic acid probes of claim 33 wherein the solid support is glass or plastic.

40. The array of nucleic acid probes of claim 33 wherein the solid support is a multi-well plate.

41. A method for detecting one or more target sequences on one or more target nucleic acids comprising:

(a) contacting one or more of the nucleic acid probes of claim 1 with a sample suspected of containing the one or more target nucleic acids; and

(b) detecting whether one or more of the first probe region and the second probe region binds their respective target sequence.

42. The method of claim 41 further comprising:

(c) amplifying the nucleic acids in the sample suspected of containing the one or more target nucleic acids prior to (a).

43. The method of claim 42 further comprising:

(d) obtaining a nucleic acid sample from a subject suspected of having a target nucleic acid prior to (c).

44. A kit for detecting one or more target sequences on one or more target nucleic acids comprising one or more of the nucleic acid probes of claim 1 and instructions for detecting one or more target sequences on one or more target nucleic acids using the one or more nucleic acid probes.

45. The kit of claim 44 further comprising one or more reagents for detecting the one or more target sequences.