METHODS FOR MOLECULAR DETECTION

• US 20120015359A1
• Filed: 09/25/2011
• Published: 01/19/2012
• Est. Priority Date: 06/06/2008
• Status: Active Grant

• Current Assignee: BioTex Incorporated
• Original Assignee: BioTex Incorporated
• Inventors: Jackson, George, Quan, Christopher
• Granted Patent: US 9,689,023 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2537/163 blocking probe
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

This invention relates to methods for molecular detection, particularly to methods utilizing target-specific molecular probes. In exemplary embodiments, target-specific molecular probes include single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) aptamers. In general, the molecular probe may bind with relatively high specificity to a given target. In one aspect, a method for molecular detection comprises a molecular probe paired to a reporter molecule wherein the molecular probe impairs the amplification of the reporter molecule in the absence of the target molecule.

Claims

1. A method for molecular detection comprising:

adding at least one molecular probe to a sample for detecting the presence of a target in said sample, said at least one molecular probe comprising an aptamer having a binding affinity for the target;

adding at least one reporter molecule to said sample, said at least one reporter molecule comprising a nucleic acid;

adding at least one primer to said sample; and

performing a quantitative or semi-quantitative amplification reaction on the at least one reporter molecule by primer extension of said primer;

wherein said at least one molecular probe binds to a conjugate region of said at least one reporter molecule in the absence of the target and the binding impairs amplification of said at least one reporter molecule by attaching to said at least one primer.

2. The method of claim 1, wherein said at least one molecular probe is added prior to the addition of either of said at least one reporter, primer or both.

3. The method of claim 2, wherein said at least one molecular probe, at least one reporter and at least one primer are added simultaneously.

4. The method of claim 1, wherein said attaching of said at least one molecular probe to said at least one primer comprises covalent linkage.

5. The method of claim 1, wherein said reporter molecule is a circular nucleic acid.

6. The method of claim 5, wherein said amplification reaction comprises rolling circle amplification.

7. The method of claim 6, further comprising performing said rolling circle amplification utilizing at least one primer substantially complementary to at least a portion of said reporter and at least a portion of its sequence is in common with said molecular probe.

8. The method of claim 7, wherein said rolling circle amplification is performed with a polymerase enzyme having strand displacement activity.

9. A method for molecular detection comprising:

adding at least one molecular probe to a sample for detecting the presence of a target in the sample, said at least one molecular probe comprising a nucleic acid and having a binding affinity for said target and being non-extensible by a nucleic acid amplification reaction;

adding at least one reporter molecule to said sample, said at least one reporter molecule comprising a nucleic acid having a first region substantially complementary to said at least one molecular probe and a second region adjacent to said first region;

adding at least one primer to said sample, said at least one primer comprising a nucleic acid and being substantially complementary to said second region;

performing a ligation reaction on said molecular probe and said primer which are bound to said first and second regions, respectively, of said reporter molecule; and

performing a quantitative or semi-quantitative nucleic acid amplification reaction on said at least one reporter molecule by primer extension of said at least one primer bound to said second region;

wherein said at least one molecular probe binds competitively to said target and said first region, said binding to said first region blocks primer extension of said at least one primer.

10. The method of claim 9, wherein said first region is upstream (5′

) of said second region.

11. The method of claim 9, further comprising sequestering any target:

molecular probe complexes prior to performing said ligation reaction.

12. The method of claim 9, wherein said reporter molecule is a circular nucleic acid.

13. The method of claim 12, wherein said amplification reaction comprises rolling circle amplification.

14. The method of claim 9, wherein said ligation reaction comprises adding a ligase to said sample.

15. The method of claim 14, wherein said ligase is selected from the group comprising E. coli DNA ligase, T4 DNA ligase, T4 RNA ligase, and a strand break DNA repair enzyme.

16. The method of claim 9, wherein said ligation reaction comprises photo-ligation.

17. A system for detecting a target molecule comprising:

at least one molecular probe comprising an aptamer having a specific binding affinity for said target molecule, said aptamer comprising a 3′

-terminus which is non-extensible by PCR;

at least one primer comprising a single-stranded nucleic acid;

at least one reporter molecule comprising a single-stranded nucleic acid having a first region substantially complementary to said at least one molecular probe and a second region substantially complementary to said at least one primer, said first and second regions being proximal to each other.

18. The system of claim 17, further comprising at least one nucleic acid polymerase.

19. The system of claim 17, wherein said first and second regions are adjacent.

20. The system of claim 19, further comprising at least one ligase.