INACTIVATABLE TARGET CAPTURE OLIGOMERS FOR USE IN THE SELECTIVE HYBRIDIZATION AND CAPTURE OF TARGET NUCLEIC ACID SEQUENCES

• US 20090286249A1
• Filed: 05/13/2009
• Published: 11/19/2009
• Est. Priority Date: 05/13/2008
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: BECKER, Michael M., LIVEZEY, Kristin W.
• Granted Patent: US 10,316,352 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

The present invention provides compositions, kits and methods for the selective hybridization and capture of a specific target nucleic acid. The specific target nucleic acid may be present in a heterogeneous mixture of nucleic acids. Selective hybridization and capture provides a target nucleic acid that is substantially free of non-target and/or contaminating nucleic acids. Target nucleic acids that have been selectively hybridized and captured using the current invention are then used in subsequent analysis, wherein the presence of non-target and/or contaminating nucleic acids that interfere with said subsequent analysis have been substantially reduced or eliminated, thereby providing improved analysis results. The invention offers the further advantage of requiring less stringent purification and/or sterility efforts than conventionally needed in order to ensure that enzymes and other reagents used in subsequent analysis, or present in the environment in which an assay is performed, are free of bacterial or other contaminating nucleic acids.

Claims

1. A preannealing reaction mixture consisting essentially of an inactivatable target capture oligomer comprising at least a binding pair member, a target hybridizing region comprising a nucleotide sequence of at least 15 to 30 nucleobases in length and a tag-closing region comprising a nucleotide sequence of at least 3 to 20 nucleobases in length, wherein said binding pair member, said target hybridizing region and said tag-closing region are joined as a single molecule, and wherein said tag-closing region nucleotide sequence is substantially complementary to a sequence contained within said target hybridizing region.

2. The reaction mixture of claim 1, wherein said tag-closing region comprises a nucleotide sequence that contains one or more nucleotide residues selected from the group consisting of:

an abasic nucleotide residue, a wobble nucleotide residue, a mismatched nucleotide residue relative to said residue'"'"'s corresponding position in said sequence contained within said target hybridizing region, and combinations thereof.

3. The reaction mixture of claim 1, wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 14 nucleobases in length.

4. The reaction mixture of claim 1, wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 9 nucleobases in length.

5. The reaction mixture of claim 1, wherein said tag-closing region comprises a nucleotide sequence of 7 nucleobases in length.

6. The reaction mixture of claim 1, wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 9 nucleobases in length and said target hybridizing regions comprises a nucleotide sequence of 17 nucleobases in length.

7. The reaction mixture of claim 1, wherein said tag-closing region is joined to an end of said target hybridizing region that is opposite the end of said target hybridizing region to which said binding pair member is joined.

8. The reaction mixture of claim 7, wherein said tag-closing region is joined to said end of said target hybridizing region using a non-nucleotide linker.

9. The reaction mixture of claim 1, wherein said target hybridizing region is joined to an end of said tag-closing region that is opposite the end of said tag-closing region to which said binding pair member is joined.

10. The reaction mixture of claim 9, wherein said target hybridizing region is joined to said end of said tag-closing region using a non-nucleotide linker.

11. The reaction mixture of claim 9, wherein said target hybridizing region is joined to said end of said tag-closing region using a nucleotide linker thereby forming a contiguous nucleotide sequence consisting essentially of said target hybridizing region and said tag-closing region.

12. The reaction mixture of claim 11, wherein said binding pair member is a substantially homopolymeric nucleotide sequence and is joined to said end of said tag-closing region using a nucleotide linker thereby forming a contiguous nucleotide sequence consisting essentially of said binding pair member, said tag-closing region and said target hybridizing region.

13. The reaction mixture of claim 12, wherein said inactivatable target capture oligomer has a 3′

to 5′

orientation such that the 5′

end of said binding pair member is joined to the 3′

end of said tag-closing region and the 5′

end of said tag-closing region is joined to the 3′

end of said target hybridizing region.

14. The reaction mixture of claim 1, further comprising at least one amplification oligomer.

15. The reaction mixture of claim 14, wherein said at least one amplification oligomer is a heterologous amplification oligomer.

16. The reaction mixture of claim 1, further comprising a set of conditions biasing said target hybridizing region towards hybridizing with a target nucleic acid and not towards hybridizing with said tag-closing region.

17. The reaction mixture of claim 1, further comprising a set of conditions biasing said target hybridizing region towards hybridizing with said tag-closing region and not towards hybridizing with a non-target nucleic acid.

18. A method for the specific hybridization and capture of a target nucleic acid, said method comprising the steps of:

a. treating a target nucleic acid sequence in a nucleic acid sample with an inactivatable target capture oligomer, wherein said inactivatable target capture oligomer comprises a target hybridizing region that is from about 15 contiguous nucleobases in length to about 30 contiguous nucleobases in length, a binding pair member and a tag-closing region, wherein said tag-closing region is from about 3 contiguous nucleobases in length to about 20 contiguous nucleobases in length, is substantially complementary to a portion of said target hybridization region, and does not stably hybridize to said target nucleic acid, and wherein said target hybridizing region, said binding pair member and said tag-closing region are joined as a single molecule;

b. providing a set of conditions wherein the stringency of said conditions biases said target hybridizing region towards stably hybridizing with said target nucleic acid and not towards stably hybridizing with said tag-closing region;

c. providing a change in said set of conditions wherein said stringency is reduced, allowing inactivatable target capture oligomer that is not stably hybridized with a target nucleic acid to form an inactive configuration by hybridizing said tag-closing region with said target hybridizing region; and

d. performing a capture step wherein a complex comprising said inactivatable capture oligomer stably hybridized with said target nucleic acid in step b is captured.

19. The method of claim 18, wherein step d. comprises providing a solid support material consisting essentially of a complementary binding pair member;

and providing conditions that allow for binding of said binding pair member of said inactivatable capture probe with said complementary binding member.

20. The method of claim 19, wherein said solid support material is a paramagnetic bead and wherein said complementary binding pair member is covalently attached to said paramagnetic bead.

21. The method of claim 20, wherein said complementary binding pair member is an immobilized probe and said binding pair member of said inactivatable capture probe is a polynucleotide region consisting essentially of a nucleotide sequence that is substantially complementary to said immobilized probe.

22. The method of claim 19, further comprising a washing step wherein said captured target nucleic acid from step d. is retained while one or more of non-captured nucleic acids, non-target nucleic acids, contaminating nucleic acids, inactivated inactivatable target capture oligomer, reagents and sample debris are separated from said captured target nucleic acid.

23. The method of claim 19, wherein said solid support material is an amine, imine or guanidine coated paramagnetic bead.

24. The method of claim 18, wherein said binding pair member of said inactivatable target capture oligomer is a polynucleotide region consisting essentially of a nucleotide sequence.

25. The method of claim 18, wherein said tag-closing region comprises a nucleotide sequence that contains one or more nucleotide residues selected from the group consisting of:

an abasic nucteotide residue, a wobble nucleotide residue, a mismatched nucleotide residue relative to said residue'"'"'s corresponding position in said sequence contained within said target hybridizing region, and combinations thereof.

26. The method of claim 18, wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 14 nucleobases in length.

27. The method of claim 18, wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 9 nucleobases in length.

28. The method of claim 18, wherein said tag-closing region comprises a nucleotide sequence of 7 nucleobases in length.

29. The method of claim 18, wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 9 nucleobases in length and said target hybridizing region comprises a nucleotide sequence of 17 nucleobases in length.

30. The method of claim 18, wherein said tag-closing region is joined to an end of said target hybridizing region that is opposite the end of said target hybridizing region to which said binding pair member is joined.

31. The method of claim 30, wherein said tag-closing region is joined to said end of said target hybridizing region using a non-nucleotide linker.

32. The method of claim 18, wherein said target hybridizing region is joined to an end of said tag-closing region that is opposite the end of said tag-closing region to which said binding pair member is joined.

33. The method of claim 32, wherein said target hybridizing region is joined to said end of said tag-closing region using a non-nucleotide linker.

34. The method of claim 32, wherein said target hybridizing region is joined to said end of said tag-closing region using a nucleotide linker thereby forming a contiguous nucleotide sequence consisting essentially of said target hybridizing region and said tag-closing region.

35. The method of claim 34, wherein said binding pair member is a substantially homopolymeric nucleotide sequence and is joined to said end of said tag-closing region using a nucleotide linker thereby forming a contiguous nucleotide sequence consisting essentially of said binding pair member, said tag-closing region and said target hybridizing region.

36. The method of claim 35, wherein said inactivatable target capture oligomer has a 3′

to 5′

orientation such that the 5′

end of said binding pair member is joined to the 3′

end of said tag-closing region and the 5′

end of said tag-closing region is joined to the 3′

end of said target hybridizing region.

37. The method of claim 18, further comprising the step of performing an analysis of said captured target nucleic acid.

38. The method of claim 37, wherein said analysis is an amplification and detection reaction.

39. The method of claim 38, wherein said amplification reaction comprises a heterologous amplification oligomer configured for generating a first amplification product containing a tag sequence, and comprises a second amplification oligomer configured to hybridize to said tag sequence or complement thereof in said first amplification product for generating subsequent amplification product.

40. The method of claim 38, wherein said detection is a nucleotide probe-based detection.

41. A method for the selective amplification of a target nucleic acid in a mixture of nucleic acids comprising target nucleic acid and one or both of non-target nucleic acid and contaminating nucleic acids, said method comprising the steps of:

a. selectively hybridizing and capturing a target nucleic acid from a nucleic acid sample using the method of claim 18;

b. performing an amplification reaction to generate a first amplification product containing a tag sequence;

c. performing a subsequent amplification reaction, wherein said subsequent amplification reaction mixture comprises an amplification oligomer configured to hybridize to said tag sequence, or complement thereof, contained within said first amplification product, to thereby generate subsequent amplification products; and

d. detecting said subsequent amplification product to determine the presence or absence of said target nucleic acid.

42. The method of claim 41, wherein said tag sequence is introduced into said first amplification product by a heterologous amplification oligomer.

43. The method of claim 41, wherein said tag sequence is introduced into said first amplification product by using said inactivatable target capture oligomer as a primer.

44. The method of claim 43, wherein said inactivatable target capture oligomer further comprises a tag region, wherein said tag region nucleotide sequence is a separate nucleotide sequence from said tag-closing region nucleotide sequence.

45. The method of claim 43, wherein said inactivatable target capture oligomer further comprises a tag region, wherein said tag region nucleotide sequence partially overlaps with said tag-closing region nucleotide sequence.

46. The method of claim 43, wherein said inactivatable target capture oligomer further comprises a tag region, wherein said tag region nucleotide sequence is the same as said tag-closing region nucleotide sequence.

47. The method of claim 41, wherein step c amplification reaction is performed in the presence of one or both of a non-target nucleic acid and a contaminating nucleic acid, and wherein substantially no non-target nucleic acid and substantially no contaminating nucleic acid contain an incorporated tag sequence from step b.

48. The method of claim 47, wherein said target nucleic acid is detected in a probe-based detection step.

49. The method of claim 41, wherein said target nucleic acid is detected in a probe-based detection step

50. The method of claim 41, wherein said tag-closing region comprises a nucleotide sequence that contains one or more nucleotide residues selected from the group consisting of:

an abasic nucleotide residue, a wobble nucleotide residue, a mismatched nucleotide residue relative to said residue'"'"'s corresponding position in said sequence contained within said target hybridizingregion, and combinations thereof.

51. The method of claim 41, wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 14 nucleobases in length.

52. The method of claim 41, wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 9 nucleobases in length.

53. The method of claim 41, wherein said tag-closing region comprises a nucleotide sequence of 7 nucleobases in length.

54. The method of claim 41, wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 9 nucleobases in length and said target hybridizing region comprises a nucleotide sequence of 17 nucleobases in length.

55. The method of claim 41, wherein said tag-closing region is joined to an end of said target hybridizing region that is opposite the end of said target hybridizing region to which said binding pair member is joined.

56. The method of claim 55, wherein said tag-closing region is joined to said end of said target hybridizing region using a non-nucleotide linker.

57. The method of claim 41, wherein said target hybridizing region is joined to an end of said tag-closing region that is opposite the end of said tag-closing region to which said binding pair member is joined.

58. The method of claim 57, wherein said target hybridizing region is joined to said end of said tag-closing region using a non-nucleotide linker.

59. The method of claim 57, wherein said target hybridizing region is joined to said end of said tag-closing region using a nucleotide linker thereby forming a contiguous nucleotide sequence consisting essentially of said target hybridizing region and said tag-closing region.

60. The method of claim 59, wherein said binding pair member is a substantially homopolymeric nucleotide sequence and is joined to said end of said tag-closing region using a nucleotide linker thereby forming a contiguous nucleotide sequence consisting essentially of said binding pair member, said tag-closing region and said target hybridizing region.

61. The method of claim 60, wherein said inactivatable target capture oligomer has a 3′

to 5′

orientation such that the 5′

end of said binding pair member is joined to the 3′

end of said tag-closing region and the 5′

end of said tag-closing region is joined to the 3′

end of said target hybridizing region.