Compositions and methods for analyzing immobilized nucleic acids
The present invention provides methods of detecting a nucleic acid analyte in a sample. 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.
1. A method of analyzing a single double-stranded nucleic acid in a sample, the method comprising:
immobilizing nucleic acids on an insoluble support in a substantially elongated configuration;
subjecting the immobilized nucleic acids to restriction endonuclease digestion, thereby creating gaps along the backbone of each double stranded nucleic acid; and
measuring the distance between the gaps along the backbone of a single nucleic acid using scanning probe microscopy, thereby obtaining a restriction profile for the nucleic acid.
2. The method of claim 1 , wherein the scanning probe microscopy is atomic force microscopy (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 the nucleic acids are modified with a methylation agent prior to restriction endonuclease digestion.
5. The method of claim 1 , wherein the nucleic acids have a length of from about 500 nucleotides to about 5000 nucleotides.
6. The method of claim 1 , wherein the nucleic acid is present in an abundance of from about 1 nucleic acid molecule per 10 2 nucleic acid molecules to about 1 nucleic acid molecule per 10 6 nucleic acid molecules.
7. The method of claim 1 , wherein the sample comprises from about 10 2 to about 10 8 distinct nucleic acids.
8. The method of claim 1 , wherein said insoluble support is planar.
9. The method of claim 1 , further comprising comparing the obtained restriction profile with a known restriction profile of a reference nucleic acid.
10. The method of claim 2 , wherein the AFM is programmed to scan the surface of the insoluble support at a higher speed than the speed used to scan the modified immobilized nucleic acids.
11. 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 a cross-sectional peak of the modified immobilized nucleic acid.
12. The method of claim 1 , further comprising comparing two obtained restriction profiles with one another.
13. The method of claim 1 , further comprising comparing three or more restriction profiles with one another.
14. The method of claim 1 , wherein the restriction profile is a binary digital profile.