System and method for inhibiting the decryption of a nucleic acid probe sequence used for the detection of a specific nucleic acid

• US 20060073487A1
• Filed: 10/01/2004
• Published: 04/06/2006
• Est. Priority Date: 10/01/2004
• Status: Active Grant

• Current Assignee: Radix BioSolutions Ltd.
• Original Assignee: Radix BioSolutions Ltd.
• Inventors: Oliver, Kerry G., Weiss, Christy, Godbout, Rebecca
• Granted Patent: US 7,892,731 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• 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

Abstract

Sequence-specific nucleic acid hybridization assays are used for the detection of specific genetic sequences as indicators of genetic anomalies, mutations, and disease propensity. In addition, they are used for the detection of various biological agents and infectious pathogens. Because a complementary probe or nucleic acid sequence is required to detect a sequence of interest in a hybridization-based assay, nucleic acid sequencing techniques can rapidly determine the specific probe sequence being used for detection. This allows reverse engineered assays to be produced rapidly. In addition, it enables the circumvention of hybridization-based assays for biological agent or infectious pathogen detection by providing the information necessary to create or alter nucleic acid sequences to produce false positives or false negatives. The present invention provides methods and compositions for inhibiting the identification of specific detection sequences. More specifically, the invention provides masking sequences that mask the identity of specific detection sequences.

Claims

1. A method of masking the identity of a detection sequence on a nucleic acid molecule comprising:

(a) obtaining a detection nucleic acid molecule comprising a known detection sequence; and

(b) associating with said nucleic acid molecule a first masking nucleic acid molecule comprising a first masking sequence, wherein the masking sequence varies from the detection sequence at one or more positions and thereby masks the identity of the detection sequence.

2. The method of claim 1, wherein the sequence of the first masking nucleic acid molecule varies from the detection sequence at two or more positions.

3. The method of claim 2, wherein the masking sequence of the first masking nucleic acid molecule varies from the detection sequence at at least half of the positions.

4. The method of claim 3, wherein the masking sequence of the first masking nucleic acid molecule varies from the detection sequence at all positions.

5. The method of claim 1, further comprising associating with said detection nucleic acid molecule and said first masking nucleic acid molecule a second masking nucleic acid molecule, wherein the second masking nucleic acid molecule comprises a second masking sequence that varies from the detection sequence and the first masking sequence at one or more positions.

6. The method of claim 5, wherein the second masking sequence varies from the detection sequence and the first masking sequence at two or more positions.

7. The method of claim 6, wherein the second masking sequence varies from the detection sequence and the first masking sequence at at least half of the positions.

8. The method of claim 7, wherein the second masking sequence varies from the detection sequence and the first masking sequence at all positions.

9. The method of claim 5, further comprising associating with said detection nucleic acid molecule and said first and second masking nucleic acid molecules a third masking nucleic acid molecule, wherein the third masking nucleic acid molecule comprises a third masking sequence that varies from the detection sequence and the first and second masking sequences at one or more positions.

10. The method of claim 9, wherein the third masking sequence varies from the detection sequence and the first and second masking sequences at two or more positions.

11. The method of claim 10, wherein the third masking sequence varies from the detection sequence and the first and second masking sequences at at least half of the positions.

12. The method of claim 11, wherein the third masking sequence varies from the detection sequence and the first and second masking sequences at all positions.

13. The method of claim 1, wherein the detection sequence is comprised within a hybridization probe.

14. The method of claim 13, wherein the hybridization probe is labeled.

15. The method of claim 14, wherein the label is biotin.

16. The method of claim 1, wherein the detection sequence is comprised within a primer or primer set.

17. The method of claim 16, wherein the primer or primer set is labeled.

18. The method of claim 17, wherein the label is biotin.

19. The method of claim 17, wherein the label is a fluorescent molecule.

20. The method of claim 19, wherein the primer or primer set further comprises a fluorescence quencher.

21. The method of claim 1, wherein the nucleotide sequence of the masking sequence is determined using an algorithm.

22. The method of claim 21, wherein for each nucleotide position of the detection sequence a complementary nucleotide is positioned in the masking sequence using the algorithm:

Masking Sequence 1 contains complementary nucleotides at nucleotide position 1 and every third nucleotide thereafter, Masking Sequence 2 contains complementary nucleotides at nucleotide position 2 and every third nucleotide thereafter, and Masking Sequence 3 contains complementary nucleotides at nucleotide position 3 and every third nucleotide thereafter.

23. The method of claim 1, wherein the detection nucleic acid molecule and the masking molecule(s) are combined on a solid support.

24. The method of claim 23, wherein the solid support is a bead.

25. The method of claim 23, wherein the solid support is a column.

26. The method of claim 1, wherein the detection nucleic acid molecule and the masking nucleic acid molecule further comprise an equilibrium sequence.

27. The method of claim 1, wherein the detection sequence and the masking sequence are from 10 to 60 nucleotides in length.

28. A method of performing a hybridization reaction comprising the steps of:

(a) obtaining a hybridization probe, which comprises a hybridization sequence designed to hybridize to a target sequence;

(b) providing a first masking nucleic acid molecule that comprises a first masking sequence, wherein the masking sequence of the first masking nucleic acid molecule varies from the hybridization sequence at one or more positions, and further wherein the first masking nucleic acid molecule has a reduced ability or inability to hybridize to the target;

(c) performing a hybridization reaction comprising the hybridization probe and the first masking nucleic acid molecule in the presence of the target sequence under conditions permitting the selective hybridization of the hybridization probe to the target sequence.

29. The method of claim 28, wherein the masking sequence of the first masking nucleic acid molecule varies from the hybridization sequence at two or more positions.

30. The method of claim 29, wherein the masking sequence of the first masking nucleic acid molecule varies from the hybridization sequence at at least half of the positions.

31. The method of claim 30, wherein the masking sequence of the first masking nucleic acid molecule varies from the hybridization sequence at all positions.

32. The method of claim 28, comprising providing a second masking nucleic acid molecule comprising a second masking sequence that varies from the hybridization sequence and the first masking sequence at one or more positions, wherein the second masking nucleic acid molecule has a reduced ability or inability to hybridize to the target.

33. The method of claim 32, wherein the second masking sequence varies from the hybridization sequence and the first masking sequence at two or more positions.

34. The method of claim 33, wherein the second masking sequence varies from the hybridization sequence and the first masking sequence at all positions.

35. The method of claim 32, comprising providing a third masking nucleic acid molecule comprising a third masking sequence that varies from the hybridization sequence and the first and second masking sequences at one or more positions, wherein the third masking nucleic acid molecule has a reduced ability or inability to hybridize to the target.

36. The method of claim 35, wherein the third masking sequence varies from the hybridization sequence and the first and second masking sequences at two or more positions.

37. The method of claim 36, wherein the third masking sequence varies from the hybridization sequence and the first and second masking sequences at all positions.

38. The method of claim 28, wherein the detection sequence and the masking sequence are from 10 to 60 nucleotides in length.

39. The method of claim 28, wherein the hybridization probe and the masking nucleic acid molecule further comprise an equilibrium sequence.

40. A composition comprising:

(a) a detection nucleic acid molecule comprising a known detection sequence; and

(b) a first masking nucleic acid molecule comprising a first masking sequence, wherein the masking sequence varies from the detection sequence at one or more positions and thereby masks the identity of the detection sequence.

41. The composition of claim 40, wherein the sequence of the first masking nucleic acid molecule varies from the detection sequence at two or more positions.

42. The composition of claim 41, wherein the masking sequence of the first masking nucleic acid molecule varies from the detection sequence at at least half of the positions.

43. The composition of claim 42, wherein the masking sequence of the first masking nucleic acid molecule varies from the detection sequence at all positions.

52. The composition of claim 40, wherein the detection sequence is comprised within a hybridization probe.

53. The composition of claim 52, wherein the hybridization probe is labeled.

54. The composition of claim 52, wherein the label is biotin.

55. The composition of claim 40, wherein the detection sequence is comprised within a primer or primer set.

56. The composition of claim 55, wherein the primer or primer set is labeled.

57. The method of claim 56, wherein the label is biotin.

58. The composition of claim 56, wherein the label is a fluorescent molecule.

59. The composition of claim 55, wherein the primer or primer set further comprises a fluorescence quencher.

60. The composition of claim 40, wherein the nucleotide sequence of the masking sequences is determined using an algorithm.

61. The composition of claim 60, wherein for each nucleotide position of the detection sequence a complementary nucleotide is positioned in the masking sequence(s) using the algorithm:

Masking Sequence 1 contains complementary nucleotides at nucleotide position 1 and every third nucleotide thereafter, Masking Sequence 2 contains complementary nucleotides at nucleotide position 2 and every third nucleotide thereafter, and Masking Sequence 3 contains complementary nucleotides at nucleotide position 3 and every third nucleotide thereafter.

62. The composition of claim 40, wherein the detection nucleic acid molecule and the masking molecule(s) are combined on a solid support.

63. The composition of claim 62, wherein the solid support is a bead.

64. The composition of claim 62, wherein the solid support is a column.

65. The composition of claim 40, wherein the detection nucleic acid molecule and the masking nucleic acid molecule further comprise an equilibrium sequence.

44. The composition of 36, further comprising a second masking nucleic acid molecule, wherein the second masking nucleic acid molecule comprises a second masking sequence that varies from the detection sequence and the first masking sequence at one or more positions.

45. The composition of claim 44, wherein the second masking sequence varies from the detection sequence and the first masking sequence at two or more positions.

46. The composition of claim 45, wherein the second masking sequence varies from the detection sequence and the first masking sequence at at least half of the positions.

47. The composition of claim 46, wherein the second masking sequence varies from the detection sequence and the first masking sequence at all positions.

48. The composition of claim 44, further comprising a third masking nucleic acid molecule, wherein the third masking nucleic acid molecule comprises a third masking sequence that varies from the detection sequence and the first and second masking sequences at one or more positions.

49. The composition of claim 48, wherein the third masking sequence varies from the detection sequence and the first and second masking sequences at two or more positions.

50. The composition of claim 49, wherein the third masking sequence varies from the detection sequence and the first and second masking sequences at at least half of the positions.

51. The composition of claim 50, wherein the third masking sequence varies from the detection sequence and the first and second masking sequences at all positions.