Method for nucleic acid sequencing

• US 20040038261A1
• Filed: 04/28/2003
• Published: 02/26/2004
• Est. Priority Date: 11/17/2000
• Status: Active Grant

• Current Assignee: Nagayama IP Holdings LLC (Toyota Tsusho Corporation)
• Original Assignee: Nagayama IP Holdings LLC (Toyota Tsusho Corporation)
• Inventors: Nagayama, Kuniaki
• Granted Patent: US 7,419,833 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: Y10S 436/801 Electron dense compounds, e.g. ferritin
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for determining the sequence of at least a portion of a single-stranded nucleic acid molecule by base-specifically labeling the exposed bases of the nucleic acid molecule using heavy element-substituted nucleotide bases which form Watson-Crick type base-pairs with the exposed bases of the nucleic acid molecule and then imaging the labeled single-stranded nucleic acid molecule using electron microscopy, e.g., transmission electron microscopy (TEM), or some other method that permits discrimination of the heavy element substituted nucleotide bases is described. The image is analyzed to determine the base sequence of at least a portion of the nucleic acid molecule.

Claims

1. A method for determining the sequence of a plurality of bases in a nucleic acid molecule, comprising:

(a) providing a support surface bearing a single stranded nucleic acid molecule comprising a plurality of bases labeled with one of four different base-specific labels;

(b) detecting the base-specifically labeled bases by electron microscopy; and

(c) analyzing the detected base-specifically labeled bases to determine the sequence of the nucleic acid molecule.

2. The method of claim 1 wherein each base-specific label comprises at least one atom having an atomic number greater than 25.

3. The method of claim 1 wherein each bases is labeled with one of four base-specific labels, wherein each of said four base-specific labels comprises a different element having an atomic weight greater than 25 and each element having an atomic weight greater than 25 differs in atomic weight from each other element having an atomic weight greater than 25 by an atomic weight of at least 15.

4. The method of claim 2 wherein the elements are selected from the group consisting of:

Pt, Eu, Pd, Co, U, Os, Fe, Hg, Gd, Cd, Zn, Ac, W, Mo, and Mn.

5. The method of claim 1 wherein the four base-specific labels consist of:

(i) a label comprising a substituted adenine,

(ii) a label comprising a substituted uracil or a substituted thymine,

(iii) a label comprising a substituted cytosine, and

(iv) a label comprising a substituted guanine.

(substitution doesn'"'"'t alter Watson-crick base pairing)

6. The method of claim 3 wherein the four elements are:

(i) Pt, Eu, Pd, and Co;

(ii) U, Os, Pd and Fe;

(iii) Hg, Gd, Cd and Zn;

or (iv) Ac, W, Mo and Mn.

7. The method of claim 5 wherein N7 or N9 of the substituted adenine is substituted with a group comprising an element having an atomic weight greater than 25.

8. The method of claim 5 wherein N7 or N9 of the substituted guanine is substituted with a group comprising an element having an atomic weight greater than 25.

9. The method of claim 5 wherein N1 of the substituted cytosine is substituted with a group comprising an element having an atomic weight greater than 25.

10. The method of claim 5 wherein N1 of the substituted uracil is substituted with a group comprising an element having an atomic weight greater than 25.

11. The method of claim 5 wherein N1 of the substituted thymnine is substituted with a group comprising an element having an atomic weight greater than 25.

12. The method of claim 5 wherein the substituted adenine has a substitution that increases the specificity of Watson-Crick type base-pairing with uracil or thymine.

13. The method of claim 5 wherein the substituted thymine or substituted uracil has a substitution that increases the specificity of Watson-Crick type base-pairing with adenine.

14. The method of claim 5 wherein the substituted cytosine has a substitution that increases the specificity of Watson-Crick type base-pairing with guanine.

15. The method of claim 5 wherein the substituted guanine has a substitution that increases the specificity of Watson-Crick type base-pairing with cytosine.

16. The method of claim 12 wherein the substituted adenine is substituted at C2 or C8 or both C2 and C8.

17. The method of claim 13 wherein the substituted thymine or the substituted uracil has a substitution at C5 or C6 or both C5 and C6.

18. The method of claim 14 wherein the substituted cytosine or the substituted uracil has a substitution at C5 or C6 or both C5 and C6.

19. The method of claim 12 wherein the substituted guanine is substituted at C2.

20. The method of any of claims 16-19 wherein the modification is a substituted or unsubsituted alkyl group or a halogen.

21. The method of claim 1 wherein the 3′

nucleotide or the 5′

nucleotide of the nucleic acid molecule is covalently bound to a defined region of the support surface.

22. The method of claim 21 wherein the defined region of the support surface is coated with gold.

23. The method of claim 1 wherein the support surface bears a plurality of single-stranded nucleic acid molecules wherein the 3′

nucleotide or the 5′

nucleotide of each nucleic acid molecule is covalently bound to a defined region of the support surface.

24. The method of claim 23 wherein each of said plurality of single-stranded nucleic acid molecules is covalently bound to a different defined region of the support surface.

25. The method of claim 2 wherein each base-specific label comprises at least 2 atoms having an atomic number greater than 25.

26. The method of claim 25 wherein each base-specific label comprises a group selected from the group consisting of:

B₁₀I₉COOH, C₂B₁₀I₁₀, C₂B₁₀Br₁₀, C₂B₁₀Cl₁₀, C₂B₁₀F₁₀.

27. The method of claim 1 wherein in the electron microscopy is transmission electron microscopy.

28. The method of claim 27 wherein the transmission electron microscopy includes the use of a transmission electron microscope comprising a Zernike phase plate located behind the objective lens.

29. The method of claim 1 wherein the electron microscopy comprises the use of a complex electron microscope.

30. The method of claim 24 wherein the plurality of nucleic acid molecules form a regular array.

31. A method for determining the sequence of a plurality of bases in a nucleic acid molecule, comprising:

(a) providing a support surface bearing a single stranded nucleic acid molecule comprising a plurality of bases;

(b) contacting the single stranded nucleic acid molecule with four different base-specific labels in the presence of an organic solvent that is free of hydrogen bond donors and acceptors to allow non-covalent binding of the four different base-specific labels to the plurality of bases;

(c) detecting the base-specifically labeled bases by electron microscopy; and

(d) analyzing the detected base-specifically labeled bases to determine the sequence of the nucleic acid molecule.

32. A substituted adenine wherein the C2 or C8 position is substituted with a C₁-C₅ alkyl group or a halogen and the N7 or N9 position is substituted with a group comprising an element having an atomic weight greater than 25.

36. The substituted adenine of claim 32 wherein the element having an atomic weight greater than 25 is selected from the group consisting of:

Pt, Eu, Pd, Co, U, Os, Fe, Hg, Gd, Cd, Zn, Ac, W, Mo, and Mn.

33. A substituted guanine wherein the C8 position is substituted with a C₁-C₅ alkyl group or a halogen and the N7 or N9 position is substituted with a group comprising an element having an atomic weight greater than 25.

37. The substituted guanine of claim 33 wherein the element having an atomic weight greater than 25 is selected from the group consisting of:

Pt, Eu, Pd, Co, U, Os, Fe, Hg, Gd, Cd, Zn, Ac, W, Mo, and Mn.

34. A substituted uracil wherein the C5 or C6 position is substituted with a C₁-C₅ alkyl group or a halogen and the N1 position is substituted with a group comprising an element having an atomic weight greater than 25.

38. The substituted uracil of claim 34 wherein the element having an atomic weight greater than 25 is selected from the group consisting of:

Pt, Eu, Pd, Co, U, Os, Fe, Hg, Gd, Cd, Zn, Ac, W, Mo, and Mn.

35. A substituted cytosine wherein the C5 or C6 position is substituted with a C₁-C₅ alkyl group or a halogen and the N1 position is substituted with a group comprising an element having an atomic weight greater than 25.

39. The substituted cytosine of claim 35 wherein the element having an atomic weight greater than 25 is selected from the group consisting of:

Pt, Eu, Pd, Co, U, Os, Fe, Hg, Gd, Cd, Zn, Ac, W, Mo, and Mn.