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

• US 7,892,731 B2
• Filed: 10/01/2004
• Issued: 02/22/2011
• 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
• Primary Examiner(s): Nguyen; Dave
• Assistant Examiner(s): BHAT, NARAYAN KAMESHWAR
• Application Number:
  US10/957,215
• Publication Number:
  US 20060073487A1
• Time in Patent Office: 2,335 Days
• Field of Search: 435/6, 435/91.1, 536/23.1, 536/24.3, 536/24.33
• US Class Current: 435/6.16
• 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 that hybridizes to a target nucleic acid sequence of interest in a sample during a hybridization assay;

(b) designing and making a first masking nucleic acid molecule comprising a first masking sequence, wherein the first masking nucleic acid molecule is the same length as the detection nucleic acid molecule and does not hybridize to a nucleic acid sequence in the sample during the hybridization assay, and wherein the masking sequence varies from the detection sequence at at least half of its nucleic acid positions,

(c) masking the identity of the detection sequence on the detection nucleic acid molecule by combining the detection nucleic acid molecule and the first masking nucleic acid molecule on the same solid support or in the same solution if the detection nucleic acid molecule and the first masking nucleic acid molecule are not immobilized on solid supports, whereby upon nucleic acid sequence analysis the presence of both the detection nucleic acid molecule and the first masking nucleic acid molecule produce a nonsensical nucleotide sequence at at least half of the nucleic acid positions in the detection sequence.

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

3. The method of claim 1, further comprising combining with said detection nucleic acid molecule and said first masking nucleic acid molecule a second masking nucleic acid molecule on the same solid support or in the same solution, wherein the second masking nucleic acid molecule is the same length as the detection nucleic acid molecule and does not hybridize to a nucleic acid sequence in the sample during the hybridization assay, and wherein the second masking nucleic acid molecule comprises a second masking sequence that varies from the detection sequence and the first masking sequence at at least half of its nucleic acid positions.

4. The method of claim 3, wherein the second masking sequence varies from the detection sequence and the first masking sequence at all positions.

5. The method of claim 3, further comprising combining with said detection nucleic acid molecule and said first and second masking nucleic acid molecules a third masking nucleic acid molecule on the same solid support or in the same solution, wherein the third masking nucleic acid molecule is the same length as the detection nucleic acid molecule and does not hybridize to a nucleic acid sequence in the sample during the hybridization assay, and 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 at least half of its nucleic acid positions.

6. The method of claim 5, wherein the third masking sequence varies from the detection sequence and the first and second masking sequences at all positions.

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

8. The method of claim 7, wherein the hybridization probe is labeled.

9. The method of claim 8, wherein the label is biotin.

10. The method of claim 1, wherein the detection nucleic acid molecule is a primer.

11. The method of claim 10, wherein the primer is labeled.

12. The method of claim 11, wherein the label is biotin.

13. The method of claim 11, wherein the label is a fluorescent molecule.

14. The method of claim 13, wherein the primer further comprises a fluorescence quencher.

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

16. The method of claim 15, 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.

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

18. The method of claim 17, wherein the solid support is a bead.

19. The method of claim 17, wherein the solid support is a column.

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

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

22. The method of claim 1, wherein the detection nucleic acid molecule and the masking molecule(s) are combined in a solution.

23. The method of claim 1, further comprising detecting the selective hybridization of the detection sequence to the target nucleic acid sequence.

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

(a) providing a hybridization probe, which comprises a hybridization sequence designed to hybridize to a target sequence in a sample during a hybridization reaction;

(b) providing a first masking nucleic acid molecule that comprises a first masking sequence, wherein the first masking nucleic acid molecule is the same length as the hybridization probe and does not hybridize to a nucleic acid sequence in the sample during the hybridization reaction, and wherein the masking sequence of the first masking nucleic acid molecule varies from the hybridization sequence at at least half of its nucleic acid positions, and further wherein the first masking nucleic acid molecule has a reduced ability or inability to hybridize to the target;

(c) masking the identity of the hybridization sequence on the nucleic acid molecule by combining the hybridization probe and the first masking nucleic acid molecule on the same solid support or in the same solution if the hybridization probe and the first masking nucleic acid molecule are not immobilized on solid supports, whereby upon nucleic acid sequence analysis the presence of both the hybridization probe and the first masking nucleic acid molecule produce a nonsensical nucleotide sequence at at least half of the nucleic acid positions in the hybridization sequence;

(d) performing the 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.

25. The method of claim 24, wherein the masking sequence of the first masking nucleic acid molecule varies from the hybridization sequence at all positions.

26. The method of claim 24, 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 at least half of its nucleic acid positions, wherein the second masking nucleic acid molecule is the same length as the hybridization probe and does not significantly hybridize to a nucleic acid sequence in the sample during the hybridization reaction, and wherein the second masking nucleic acid molecule has a reduced ability or inability to hybridize to the target;

and combining the second masking nucleic acid molecule with the hybridization probe and the first masking nucleic acid molecule on the same solid support or in the same solution.

27. The method of claim 26, wherein the second masking sequence varies from the hybridization sequence and the first masking sequence at all positions.

28. The method of claim 26, 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 at least half of its nucleic acid positions, wherein the third masking nucleic acid molecule is the same length as the hybridization probe and does not significantly hybridize to a nucleic acid sequence in the sample during the hybridization reaction, and wherein the third masking nucleic acid molecule has a reduced ability or inability to hybridize to the target;

and combining the third masking nucleic acid molecule with the hybridization probe, the first masking nucleic acid molecule, and the second masking nucleic acid molecule on the same solid support or in the same solution.

29. The method of claim 28, wherein the third masking sequence varies from the hybridization sequence and the first and second masking sequences at all positions.

30. The method of claim 24, wherein the hybridization sequence and the masking sequence are from 10 to 60 nucleotides in length.

31. The method of claim 24, wherein the hybridization probe and the masking nucleic acid molecule further comprise an equilibrium sequence.

32. The method of claim 24, wherein the hybridization probe and the masking molecule(s) are combined in a solution.

33. The method of claim 24, wherein the hybridization probe and the masking molecule(s) are combined on a solid support.

34. The method of claim 24, further comprising detecting the selective hybridization of the hybridization probe to the target sequence.