Sequencing nucleic acid polymers with electron microscopy

• US 8,153,438 B2
• Filed: 04/02/2010
• Issued: 04/10/2012
• Est. Priority Date: 10/04/2007
• Status: Expired due to Fees

• Current Assignee: Aeon Biowares, Inc.
• Original Assignee: Halcyon Molecular
• Inventors: Andregg, William, Andregg, Michael
• Primary Examiner(s): Gakh, Yelena G
• Assistant Examiner(s): Hixson, Christopher A
• Application Number:
  US12/753,693
• Publication Number:
  US 20100267152A1
• Time in Patent Office: 739 Days
• Field of Search: 436/94, 436/149, 436/171, 435/6, 435/287.2, 702/20
• US Class Current: 436/94
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to using an electron microscope to sequence by direct inspection of labeled, stretched DNA. This method will have higher accuracy, lower cost, and longer read length than current DNA sequencing methods.

Claims

1. A method of achieving consistent base-to-base spacing in a nucleic acid polymer strand comprising:

a) introducing a portion of a polymer attachment tool into a solution comprising nucleic acid polymer strands;

b) attaching a section of at least one strand to the tool;

c) removing substantially all of the tool with the attached strand from the solution after which at least one region of the strand is suspended between the solution and the tool;

d) depositing the suspended region of the strand onto a substrate, wherein the strand detaches from the tool; and

e) obtaining consistent spacing between consecutive bases of the strand.

2. The method of claim 1, wherein the at least one suspended region is suspended between the interface of two surfaces.

3. The method of claim 1, wherein the at least one suspended region is suspended between two phases.

4. The method of claim 1, wherein the at least one suspended region is suspended between an air/solvent interface and the tool.

5. The method of claim 1, wherein the at least one suspended region is suspended between a liquid solution and a solid surface.

6. The method of claim 3, wherein the two phases are selected from the group consisting of a solid, liquid, and gas phase.

7. The method of claim 1, wherein the suspended region of the strand is straightened.

8. The method of claim 1, further comprising placing a plurality of suspended regions on a substrate wherein said suspended regions are substantially parallel to one another.

9. The method of claim 8, wherein the suspended regions do not overlap with one another on the substrate.

10. The method of claim 1, wherein the spacing between consecutive bases in the suspended region ranges from 3 angstroms to 7 angstroms.

11. The method of claim 1, wherein the tool is configured to attach to multiple strands.

12. The method of claim 1, wherein the polymer attachment tool is a needle.

13. The method of claim 12, wherein the tool is a needle array.

14. The method of claim 1, wherein the tool is capable of attaching to multiple regions on a single strand.

15. The method of claim 1, wherein the suspended region is at least 20 nanometers in length.

16. The method of claim 1, wherein the suspended region is at least 100 microns in length.

17. The method of claim 16, wherein the base pairs in the suspended region do not overlap.

18. The method of claim 1, wherein the tool manipulates the strand while the strand is in solution.

19. The method of claim 1, wherein the nucleic acid polymer strands comprise single stranded nucleic acid strands.

20. The method of claim 1, wherein the nucleic acid polymer strands comprise double stranded nucleic acid strands.

21. A method of achieving consistent base-to-base spacing in a nucleic acid strand, comprising:

a) introducing a portion of a polymer attachment tool into a solution comprising nucleic acid polymer strands;

b) attaching a section of at least one strand to the tool;

c) removing substantially all of the tool with the attached strand from the solution after which at least one region of the strand is suspended between the solution and the tool;

d) depositing the suspended region of the strand onto a substrate, wherein the substrate is distinct from the polymer attachment tool;

e) obtaining consistent spacing between consecutive bases of the strand.

22. A method of sequencing a labeled nucleic acid polymer strand, comprising:

a) introducing a portion of a polymer attachment tool into a solution comprising nucleic acid polymer strands;

b) attaching a strand to the tool;

c) removing substantially all of the tool with the attached strand from the fluid after which at least one suspended region of the strand is suspended between the fluid and the tool;

d) obtaining consistent spacing between consecutive bases in said suspended region of the strand;

e) placing the strand on a substrate wherein the substrate is distinct from the polymer attachment tool and such that the consistent spacing is substantially preserved;

f) imaging the strand with an electron microscope (EM) to produce EM data; and

g) determining the base sequence for substantially the entire strand from the EM data;

wherein at least a subset of the bases of the strand are associated with a label that is resolvable by an electron microscope.

23. The method of claim 22, wherein the label is a contrast agent label.

24. The method of claim 23, wherein the contrast agent is a high-Z-atom labeling compound.

25. The method of claim 22, wherein the label is a cluster label.

26. The method of claim 23, wherein the contrast agent directly labels the nucleic acid polymer strand.

27. The method of claim 22, wherein the bases are associated with a single label type.

28. The method of claim 22, wherein the bases are associated with a single label type under two different reaction conditions

29. The method of claim 22, wherein the bases are associated with two label types.

30. The method of claim 22, wherein the imaging step determines the location of bases associated with labels and bases not associated with labels.

31. The method of claim 22, wherein the nucleic acid polymer strand is a DNA strand.

32. The method of claim 31, wherein thymine and cytosine are selectively associated with labels while adenine and guanine are not, significantly, associated with labels.

33. The method of claim 31, wherein only the guanine bases are, significantly, associated with labels.

34. The method of claim 32, wherein only the thymine bases are, significantly, associated with labels.

35. The method of claim 32, wherein only the adenine bases are, significantly, associated with labels.

36. The method of claim 32, wherein only the uracil bases are, significantly, associated with labels.

37. The method of claim 32, wherein only the cytosine bases are, significantly, associated with labels.