Compositions and methods for analyzing immobilized nucleic acids

• US 20070092905A1
• Filed: 10/17/2006
• Published: 04/26/2007
• Est. Priority Date: 10/21/2005
• Status: Active Grant

• Current Assignee: New York University, Regents of the University of California (University of California)
• Original Assignee: New York University
• Inventors: Mishra, Bhubaneswar, Reed, Jason, Gimzewski, James
• Granted Patent: US 8,566,038 B2
• Time in Patent Office: Days
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]

Abstract

The present invention provides methods of detecting a nucleic acid analyte in a sample. The methods generally involve modifying immobilized nucleic acids from a sample onto an insoluble support in a substantially elongated configuration, where modification generates an identifying feature that identifies the analyte; and detecting the identifying feature(s) using scanning probe microscopy, to detect the analyte. The present invention further provides a method for assigning a profile of a feature to a nucleic acid. The present invention further provides a computer program product for use in a subject method. The present invention further provides a system for detecting a nucleic acid in a sample; and a system for assigning a profile of a feature to a nucleic acid. The present invention further provides a method for immobilizing a nucleic acid onto an insoluble support; and further provides insoluble support having nucleic acid(s) immobilized thereon. The present invention further provides a method of diagnosing a disorder or condition in an individual, where the method involves use of a subject method for detecting a nucleic acid analyte.

Claims

1. A method of detecting a nucleic acid analyte in a sample, the method comprising:

modifying immobilized nucleic acids from said sample with a modifying agent, wherein the immobilized nucleic acids are immobilized on an insoluble support in a substantially elongated configuration, and wherein said modification generates one or more identifying features that identify the analyte; and

detecting said one or more identifying features using scanning probe microscopy to detect the presence of said nucleic acid analyte in said sample.

2. The method of claim 1, wherein the one or more identifying features are detected using an atomic force microscope (AFM).

3. The method of claim 1, wherein the insoluble support retains nucleic acids at least about 10 base pairs in length.

4. The method of claim 1, wherein said modifying with a modifying agent comprises cleaving said immobilized nucleic acids with one or more restriction endonucleases to generate immobilized nucleic acid restriction fragments, wherein said identifying features are restriction endonuclease pattern, and wherein detecting the identifying features comprises measuring the lengths of said immobilized fragments.

5. The method of claim 1, wherein said modifying with a modifying agent comprises methylating said immobilized nucleic acids with a methylating agent, wherein said one or more identifying feature is a methylation pattern, and wherein detecting the identifying features comprises detecting lengths of immobilized fragments generated using a methylation-sensitive restriction endonuclease.

6. The method of claim 1, wherein said modifying with a modifying agent comprises contacting said immobilized nucleic acids with a nucleic acid probe, wherein said one or more identifying feature is a hybridization of the nucleic acid probe to the immobilized nucleic acid, and wherein detecting the identifying features comprises detecting a height difference between immobilized nucleic acid hybridized to the nucleic acid probe and immobilized nucleic acid not hybridized to the probe.

7. The method of claim 1, wherein said modifying with a modifying agent comprises binding with a sequence-specific nucleic acid-specific binding protein, wherein said one or more identifying feature is binding of the nucleic acid-specific binding protein to the immobilized nucleic acid, and wherein detecting the identifying features comprises detecting a height difference between immobilized nucleic acid bound to the protein and immobilized nucleic acid not bound to the protein.

8. The method of claim 1, wherein the nucleic acids have a length of from about 500 nucleotides to about 5000 nucleotides.

9. The method of claim 1, wherein the nucleic acid analyte is present in an abundance of from about 1 nucleic acid molecule per 10² nucleic acid molecules to about 1 nucleic acid molecule per 10⁶ nucleic acid molecules.

10. The method of claim 1, wherein the sample comprises from about 10² to about 10⁸ distinct nucleic acid species.

11. The method of claim 1, wherein said method is qualitative.

12. The method of claim 1, wherein said method is quantitative.

13. The method of claim 1, wherein said insoluble support is planar.

14. The method of claim 1, wherein said insoluble support is spherical.

15. The method of claim 1, further comprising comparing the detected identifying features with known identifying feature of a reference nucleic acid.

16. The method of claim 2, wherein the AFM is programmed to scan the surface of the insoluble support at a higher speed than the modified immobilized nucleic acids.

17. The method of claim 2, wherein the AFM is programmed to obtain two or more cross-sectional profile data points per unit length of modified immobilized nucleic acid, wherein the unit length is at least about 2 nanometers, wherein at least one of the data points is taken from the cross-sectional peak of the modified immobilized nucleic acid.

18. A method for assigning a profile of a feature to a nucleic acid, the method comprising:

modifying an immobilized nucleic acid with one or more nucleic acid modifying agents, wherein the immobilized nucleic acid are immobilized onto an insoluble support in a substantially elongated configuration, and wherein said modification generates a modification feature;

detecting the modification feature of the immobilized nucleic acid using atomic force microscopy; and

assigning a character to each feature, thereby generating a profile of the feature.

19. The method of claim 18, further comprising comparing the profile with a reference profile.

20. The method of claim 18, further comprising comparing two profiles with one another.

21. The method of claim 18, further comprising comparing three or more profiles with one another.

22. The method of claim 18, wherein the profile is a binary digital profile.

23. The method of claim 18, wherein the feature is a restriction endonuclease site, and the profile is a restriction endonuclease digestion pattern.

24. The method of claim 18, wherein the feature is methylation of cytosine residues, and the profile is a methylation pattern.

25. A computer program stored on a computer-readable storage medium, wherein the computer program, when read by a computer, executes calculation of a feature of a modified immobilized nucleic acid.

26. The computer program of claim 25, wherein the feature is a restriction pattern, and wherein the computer program executes calculation of the length, in base pairs, of ordered restriction fragments immobilized on an insoluble support surface, based on the contour length, in nanometers, detected by atomic force microscope.

27. The computer program of claim 25, wherein the feature is binding of a nucleic acid binding moiety to the immobilized nucleic acid, and wherein the computer program executes calculation of the location of the bound nucleic acid binding moiety on the immobilized nucleic acid.

28. The computer program of claim 27, wherein the binding moiety is selected from a nucleic acid probe, a nucleic acid binding protein, a nanoparticle, and a detectable label.

29. A system for detecting a nucleic acid in a sample, the system comprising:

an atomic force microscope (AFM), wherein the AFM detects a modification feature of a modified nucleic acid, wherein the nucleic acid is immobilized on an insoluble substrate in a substantially elongated configuration;

a central computing environment;

an input device, operatively connected to the computing environment, to receive modification feature data from the AFM; and

a computer program of claim 25 stored on a computer-readable storage medium.

30. The system of claim 29, wherein the modification feature data are communicated to an output device operatively connected to the central computing environment.

31. The system of claim 30, further comprising an insoluble substrate.

32. A computer program stored on a computer-readable storage medium, wherein the computer program, when read by a computer, assigns a profile of a feature to a test nucleic acid.

33. The computer program of claim 32, wherein the feature profile is selected from a restriction pattern, a methylation pattern, and a pattern of binding of a nucleic acid binding moiety.

34. The computer program of claim 32, wherein the computer program further compares the test feature profile to a reference feature profile.

35. The computer program of claim 32, wherein the computer program compares a first test feature profile to at least a second test feature profile.

36. A system for assigning a profile of a feature to a nucleic acid, the system comprising:

a) an atomic force microscope (AFM), wherein the AFM detects a feature of a nucleic acid,

b) a central computing environment;

c) an input device operatively connected to the computing environment, to receive nucleic acid length data from the AFM;

d) a computer program product contained within the central computing environment, wherein the computer program product assigns a profile of a feature to a nucleic acid, based on data input from the AFM.

37. A method for immobilizing a nucleic acid onto an insoluble support, the method comprising:

depositing a nucleic acid onto a surface of an insoluble support, wherein the nucleic acid is deposited in a substantially elongated configuration;

applying a shadow mask onto the deposited nucleic acid, wherein the shadow mask comprises a plurality of linear gaps; and

depositing a securing material onto the deposited nucleic acid through the gap, thereby immobilizing the deposited nucleic acid.

38. The method of claim 37, wherein the plurality of linear gaps are substantially parallel to one another.

39. The method of claim 37, wherein the gaps are substantially perpendicular to the deposited nucleic acid.

40. The method of claim 37, wherein the surface is derivatized to provide for a positively charged surface.

41. The method of claim 40, wherein the surface of the insoluble support is derivatized with 3-aminopropyl triethoxysilane.

42. The method of claim 37, wherein the gaps are from about 75 nm to about 250 nm apart.

43. The method of claim 37, wherein the gaps are from about 5 nm to about 20 nm wide.

44. The method of claim 37, wherein the insoluble support comprises mica.

45. The method of claim 37, wherein the securing material is an inert metal.

46. The method of claim 37, wherein the securing material is selected from gold, platinum, titanium, rhodium, and a nickel-cobalt alloy.

47. The method of claim 37, wherein said insoluble support is planar.

48. The method of claim 37, wherein said insoluble support is a bead.

49. The method of claim 37, wherein said insoluble support comprises a channel.

50. An insoluble support having immobilized thereon a nucleic acid, wherein the nucleic acid is immobilized onto the insoluble support by a securing agent.

51. The insoluble support of claim 50, wherein the securing agent is an inert metal.

52. The insoluble support of claim 50, wherein the nucleic acid is immobilized at more than one point by a plurality of securing agents.

53. The insoluble support of claim 50, wherein the plurality of securing agents are spaced from about 5 nm to about 250 nm apart.

54. The insoluble support of claim 50, wherein the insoluble support comprises mica.

55. The insoluble support of claim 50, wherein the nucleic acid is immobilized in a substantially elongated configuration.

56. The insoluble support of claim 50, comprising a plurality of nucleic acids immobilized thereon.

57. A method of diagnosing a disorder or condition in an individual, the method comprising:

detecting a nucleic acid analyte in a sample obtained from the individual, wherein the nucleic acid analyte is diagnostic of the disorder or condition, wherein said detecting comprises;

i) modifying immobilized nucleic acids from said sample with a modifying agent, wherein the immobilized nucleic acids are immobilized on an insoluble support in a substantially elongated configuration, and wherein said modification generates one or more identifying features that identify the analyte; and

ii) detecting an identifying feature of said immobilized nucleic acids to detect the presence of said nucleic acid analyte in said sample.

58. The method of claim 57, wherein the presence of the nucleic acid analyte is diagnostic of the disorder.

59. The method of claim 57, wherein the abundance of the nucleic acid analyte is diagnostic of the disorder.

60. A computer program stored on a computer-readable storage medium, wherein the computer program, when read by a computer, controls the movement of a scanning tip of an atomic force microscope (AFM).

61. The computer program of claim 60, wherein the computer program causes an increase in scan speed.

62. The computer program of claim 60, wherein the computer program causes the AFM tip to skip scanning of nucleic acids having a preselected exclusion criterion.