Methods and Compositions Related to Nucleic Acid Detection

• US 20090042735A1
• Filed: 03/05/2008
• Published: 02/12/2009
• Est. Priority Date: 03/05/2007
• Status: Abandoned Application

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Bishop, Justin, Chagovetz, Alexander M., Blair, Steven M.
• Application Number:
  US12/042,516
• Publication Number:
  US 20090042735A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• 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

Disclosed are compositions and a method for detection of nucleic acid sequences based on competitive displacement.

Claims

1. A method of determining sequence similarity of a target nucleic acid and a probe, the method comprising:

a) bringing into contact the target nucleic acid, the probe, and a labeled competitor nucleic acid;

b) simultaneously incubating the target nucleic acid, a probe, and the labeled competitor nucleic acid under conditions suitable for hybridization;

c) determining the binding pattern of the competitor nucleic acid to the probe in the presence of the target nucleic acid; and

d) determining sequence similarity of the target nucleic acid and the probe based on the results of step c).

4. The method of claim 1, wherein the target nucleic acid is labeled.

5. The method of claim 4, wherein the target nucleic acid label is distinct from the competitor nucleic acid label.

6. The method of claim 1, wherein the labeled competitor nucleic acid is present in higher concentrations.

7. The method of claim 1, wherein step c) is determined in real time.

8. The method of claim 7, wherein the hybridization pattern differs as a function of time.

9. The method of claim 8, wherein the labeled competitor nucleic acid initially dominates hybridization, and is then displaced by the target nucleic acid.

10. The method of claim 9, wherein the target nucleic acid and the probe are complementary in sequence.

11. The method of claim 7, wherein the target nucleic acid and the probe are not complementary in sequence.

12. The method of claim 11, wherein the target nucleic acid and the probe differ in sequence by one nucleotide.

13. The method of claim 9, wherein the labeled competitor nucleic acid and the probe are complementary in sequence

14. The method of claim 9, wherein the labeled competitor nucleic acid and the probe are not complementary in sequence.

15. The method of claim 14, wherein the labeled competitor nucleic acid and the probe differ by one nucleotide.

16. The method of claim 1, wherein the probe is attached to a solid support.

17. The method of claim 16, wherein the solid support is a microarray.

18. The method of claim 17, wherein more than one probe nucleic acid is used.

19. The method of claim 16, wherein said solid support is selected from the group consisting of glass and plastic.

20. The method of claim 1, wherein the target nucleic acid comprises a single nucleotide polymorphism (SNP).

21. The method of claim 1, wherein the labeled competitor nucleic acid molecule is fluorescently labeled.

22. The method of claim 1, wherein more than one labeled competitor nucleic acid is used.

23. The method of claim 22, wherein each labeled competitor nucleic acid is labeled with a different label.

24. The method of claim 1, wherein the probe is selected from the group consisting of DNA, RNA, or a combination thereof.

25. The method of claim 17, wherein the probes are attached to the microarray by a spacer.

26. The method of any one claim 1, wherein the target nucleic acid is selected from the group consisting of DNA, RNA, a peptide nucleic acid, or a combination thereof.

27. The method of claim 26, wherein the target DNA is cDNA.

28. The method of claim 26, wherein the DNA is mtDNA.

29. The method of claim 26, wherein the target RNA is mRNA.

30. The method of claim 26, wherein the target RNA is miRNA.

31. The method of claim 1, wherein the target is not amplified before being put in contact with the probe and the labeled competitor nucleic acid.

32. The method of claim 1, wherein the target is amplified before being put in contact with the probe and the labeled competitor nucleic acid.

33. The method of claim 1, wherein the labeled competitor nucleic acid is selected from the group consisting of DNA, RNA, a peptide nucleic acid, or a combination thereof.

35. The method of claim 1, further comprising curve fitting of the binding pattern of the labeled nucleic acid, thereby determining the sequence of the target nucleic acid.

36. The method of claim 35, wherein curve fitting of the binding patterns of the labeled nucleic acid to two or more probe sequences is used to determine the sequence of the target nucleic acid.

37. The method of claim 35, wherein curve fitting of the binding patterns of multiple competitor nucleic acid sequences is used determine the sequence similarity of the target nucleic acid and the probe

38. The method of claims 37, wherein curve fitting of the binding patterns of multiple competitor nucleic acid sequences is used to determine the sequences of two or more target nucleic acid sequences

40. The methods of claim 36, wherein the effects of binding of additional non-target nucleic acid sequences on the binding pattern of the competitor nucleic acid sequences is used to determine the sequence of the target nucleic acid.

41. The methods claim 36, wherein curve fitting of the binding patterns of the competitor nucleic acid and target nucleic acid sequences are used to determine the sequence of the target nucleic acid.

42. The method of claim 41, wherein the effects of binding of additional non-target nucleic acid sequences on the binding patterns of the competitor nucleic acid and target nucleic acid sequences is taken into account in determining the sequence of the target nucleic acid.

43. The method of claim 1, wherein determining the binding pattern of the competitor nucleic acid to the probe in the presence of the target nucleic acid occurs using real time signal acquisition.

44. The method of claim 43, wherein the real time signal acquisition is obtained using fluorescent resonance energy transfer (FRET).

45. The method of claim 43, wherein the real time signal acquisition is obtained using TIRF or evanescent wave excitation.

46. The method of claim 45, wherein evanescent wave excitation is accomplished via an optical waveguide.

47. The method of claim 46, wherein said optical waveguide is substantially planar.

48. The method of claim 47, wherein said substantially planar optical waveguide comprises thin dielectric or metallic films.

49. The method of claim 1, wherein the labeled competitor nucleic acid is labeled with nanoparticle scattering labels.

50. The method of claim 1, wherein the labeled competitor nucleic acid is labeled with Raman scattering labels.

2. A method of detecting the presence of a target nucleic acid in a sample, the method comprising:

a) simultaneously bringing into contact the sample, a probe, and a labeled competitor nucleic acid;

b) incubating the sample, the probe, and the labeled competitor nucleic acid under conditions suitable for hybridization;

c) determining the binding pattern of the competitor nucleic acid to the probe in the presence of the sample; and

d) detecting the presence of the target nucleic acid based on the results of step c).

3. A method of quantifying a target nucleic acid, the method comprising:

a) simultaneously bringing into contact a target nucleic acid, a probe, and a labeled competitor nucleic acid;

b) incubating the target nucleic acid, the probe, and the labeled competitor nucleic acid under conditions suitable for hybridization;

c) determining the amount of labeled competitor nucleic acid bound to the nucleic acid probe; and

d) quantifying the target nucleic acid based on the results of step c)