Novel nucleic acid complexes and detection thereof

• US 20050260624A1
• Filed: 02/28/2005
• Published: 11/24/2005
• Est. Priority Date: 02/28/2004
• Status: Abandoned Application

• Current Assignee: Chang Ning J. Wang
• Original Assignee: Chang Ning J. Wang
• Inventors: Wang, Chang Ning J.
• Application Number:
  US11/069,370
• Publication Number:
  US 20050260624A1
• 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 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 1/706 for hepatitis
• CPC Class Codes: C12Q 2523/113 Denaturating agents
• CPC Class Codes: C12Q 2537/119 Triple helix formation
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide

Abstract

A nucleic acid complex that contains a plurality of first nucleic acids, a plurality of second nucleic acids, and a plurality of third nucleic acids, in which each of the first nucleic acids, complementary to each of the second nucleic acids, contains a sequence which is complementary to a site of each of the third nucleic acids; the number of the first nucleic acids and that of the second nucleic acids are each 1 to 10¹² times that of the third nucleic acids; and the first, second, and third nucleic acids are crosslinked to form the nucleic acid complex. Also disclosed is a detection method in which the above-described nucleic acid complex is used as a detectable means for identifying a specific nucleic acid target site.

Claims

1. A nucleic acid complex, comprising a plurality of first nucleic acids, a plurality of second nucleic acids, and a plurality of third nucleic acids, wherein each of the first nucleic acids, complementary to each of the second nucleic acids, contains a sequence which is complementary to a site of each of the third nucleic acids;

the number of the first nucleic acids and the number of the second nucleic acids are each 1 to 10¹² times that of the third nucleic acids; and

the first nucleic acids, the second nucleic acids, the third nucleic acids are crosslinked to form the nucleic acid complex, which, when excited at 518 nm after staining with ethidium bromide, emits fluorescence at 605 nm with an intensity at least 10 times that of non-crosslinked first, second, and third nucleic acids.

2. The nucleic acid complex of claim 1, wherein the number of the first nucleic acids and the number of the second nucleic acids are each 10³ to 10⁸ times that of the third nucleic acids.

3. The nucleic acid complex of claim 2, wherein the first and second nucleic acids are each 100 to 20,000 nucleotides in length.

4. The nucleic acid complex of claim 3, wherein the first and second nucleic acids are each 200 to 8,000 nucleotides in length.

5. The nucleic acid complex of claim 4, wherein the complementary sequence is 10 to 20,000 nucleotides in length.

6. The nucleic acid complex of claim 5, wherein the complementary sequence is 20 to 8,000 nucleotides in length.

7. The nucleic acid complex of claim 2, wherein the complementary sequence is 10 to 20,000 nucleotides in length.

8. The nucleic acid complex of claim 7, wherein the complementary sequence is 20 to 8,000 nucleotides in length.

9. The nucleic acid complex of claim 3, wherein the complementary sequence is 10 to 20,000 nucleotides in length.

10. The nucleic acid complex of claim 9, wherein the complementary sequence is 20 to 8,000 nucleotides in length.

11. A process for detecting a target site in a nucleic acid, comprising:

providing a plurality of first nucleic acids, a plurality of second nucleic acids, and a plurality of third nucleic acids suspected of containing a target site, wherein each of the first nucleic acids, complementary to each of the second nucleic acids, contains a sequence which is complementary to a target site of each of the third nucleic acids; and

the number of the first nucleic acids and the number of the second nucleic acids are each 1 to 10¹⁶ times that of the third nucleic acids;

denaturing and localizing to a planar surface the first, second, and third nucleic acids, thereby facilitating formation of a crosslinked nucleic acid complex if each of the third nucleic acids contains the target site; and

detecting presence or absence of the crosslinked nucleic acid complex.

12. The process of claim 11, wherein the number of the first nucleic acids and the number of the second nucleic acids are each 10³ to 10¹³ times that of the third nucleic acids.

13. The process of claim 12, wherein the first and second nucleic acids are each 100 to 20,000 nucleotides in length.

14. The process of claim 13, wherein the first and second nucleic acids are each 200 to 8,000 nucleotides in length.

15. The process of claim 14, wherein the complementary sequence is 10 to 20,000 nucleotides in length.

16. The process of claim 15, wherein the complementary sequence is 20 to 8,000 nucleotides in length.

17. The process of claim 16, wherein the denaturation is achieved by mixing the first, second, and third nucleic acids in a chaotropic aqueous solvent.

18. The process of claim 17, wherein the localization is achieved by adding a hydrophobic organic solvent to the chaotropic aqueous solvent, the interface between the two solvents constituting the planar surface.

19. The process of claim 18, wherein the hydrophobic organic solvent is n-butylalcohol, tert-amylalcohol, cyclohexyl alcohol, phenol, p-methoxyphenol, benzyl alcohol, aniline, pyridine, purine, 3-aminotriazole, butyramide, hexamide, thioacetamide, δ

-valarolactam, tert-butylurea, ethylenethiourea, allylthiourea, thiourea, urethane, N-propylurethane, N-methylurethane, cyanoguanidine, or a combination thereof.

20. The process of claim 19, wherein the hydrophobic organic solvent is aniline.

21. The process of claim 18, wherein the chaotropic aqueous solvent contains Mg²⁺, Ca²⁺, Na⁺, K⁺, NH₄⁺, Cs⁺, Li⁺, (CH₃)₄N⁺, or a combination thereof.

22. The process of claim 18, wherein the chaotropic aqueous solvent contains tosylate⁻

, Cl₃CCOO⁻

, SCN⁻

, ClO₄⁻

, I⁻

, Br⁻

, Cl⁻

, BrO₃⁻

, CH₃COO⁻

, HSO₃⁻

, F⁻

, SO₄²⁻

, (CH₃) ₃CCOO⁻

, HPO₄⁻

, or a combination thereof.

23. The process of claim 22, wherein the chaotropic aqueous solvent contains SCN⁻

.

24. The process of claim 12, wherein the denaturation is achieved by mixing the first, second, and third nucleic acids in a chaotropic aqueous solvent.

25. The process of claim 24, wherein the localization is achieved by adding a hydrophobic organic solvent to the chaotropic aqueous solvent, the interface between the two solvents constituting the planar surface.

26. The process of claim 25, wherein the detection is achieved by determining the intensity of fluorescence emitted from the crosslinked nucleic acid complex after it is stained with a double stranded DNA intercalating fluorescent dye.