Methods for determining nucleotide sequences of single nucleic acid molecules

• US 7,223,568 B2
• Filed: 11/30/2001
• Issued: 05/29/2007
• Est. Priority Date: 03/30/2000
• Status: Expired due to Fees

• Current Assignee: Genesis Research Institute, Inc., Toyota Jidosha Kabushiki Kaisha (Toyota Motor Corporation)
• Original Assignee: Genesis Research Institute, Inc., Toyota Jidosha Kabushiki Kaisha (Toyota Motor Corporation)
• Inventors: Mafune, Fumitaka, Takeda, Yoshihiro, Kondow, Tamotsu
• Primary Examiner(s): Whisenant; Ethan
• Assistant Examiner(s): Lu; Frank Wei Min
• Application Number:
  US09/996,591
• Publication Number:
  US 20020143167A1
• Time in Patent Office: 2,006 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention relates to a method for determining a nucleotide sequence of a nucleic acid by detecting a single dye molecule.

Claims

1. A method for determining a nucleotide sequence of a single nucleic acid molecule, which comprises:

(a) immobilizing a nucleic acid molecule onto the surface of a solid;

(b) annealing a primer to said nucleic acid molecule, wherein said primer has a sequence complementary to a part of a sequence of the nucleic acid molecule;

(c) providing a solution which contains a DNA polymerase and only one type of dye-labeled dNTP, where N is A, T or U, G or C, or an RNA polymerase and only one type of dye-labeled NTP, where N is A, U, G or C, to said immobilized nucleic acid molecule, and allowing the dye-labeled dNTP or NTP to react with the 3′

end of said primer, whereby the dye-labeled dNTP or NTP, which forms a base-pair with a base in the nucleic acid molecule at a position where the dye-labeled dNTP or NTP reacts with the 3′

end of said primer and is bound to the primer by action of the polymerase;

(d) detecting a bound, dye-labeled dNTP or NTP;

(e) disrupting the dye molecule of the bound, dye-labeled dNTP or NTP;

(f) repeating (c) to (e) while changing the type of dye-labeled dNTP or NTP in turn, to sequentially bind dNTPs or NTPs which forms a base-pair with the nucleotides of the nucleic acid molecule; and

(g) determining a nucleotide sequence of the nucleic acid molecule based on the types of the sequentially bound dNTPs or NTPs.

2. The method of claim 1, wherein said surface of a solid is the inner wall of a capillary.

3. The method of claim 1, wherein (d) comprises optically detecting the dye molecule of said dye-labeled dNTP or NTP.

4. The method of claim 1, wherein (d) comprises exciting dye molecules by irradiation of a laser beam and detecting a fluorescent signal.

5. The method of claim 1, wherein said detection is performed using a confocal fluorescence microscope system.

6. The method of claim 4, wherein said disrupting the dye molecules in (e) comprises irradiating with a laser beam stronger than the laser beam in (d).

7. The method of claim 1, wherein said dye is a fluorescent dye.

8. The method of claim 1, wherein said dye-labeled dNTP is labeled with rhodamine, tetramethyl rhodamine (fluorescein) Rhodamine 6G, fluorescein isothiocyanate, or 4-fluoro-7-nitro-benzofurazon (Texas Red).

9. The method of claim 1, wherein said dye-labeled NTP is labeled with rhodamine, tetramethyl rhodamine (fluorescein) Rhodamine 6G, fluorescein isothiocyanate, or 4-fluoro-7-nitro-benzofurazon (Texas Red).

10. The method of claim 1, wherein said dNTP or NTP is each labeled with the same dye.

11. The method of claim 1, wherein said solution consists of a droplet in which an aqueous solution containing said dye-labeled dNTP or NTP, is entrapped within a hydrophobic liquid.

22. The method of claim 1, wherein said solution consists of a droplet in which an aqueous solution containing said dye-labeled dNTP or NTP, is entrapped within a hydrophobic liquid.

12. A method for determining a nucleotide sequence of a single nucleic acid molecule, which comprises:

(a) immobilizing a primer onto the surface of a solid, wherein the primer comprises a sequence complementary to a part of a sequence in the nucleic acid molecule;

(b) annealing a nucleic acid molecule to the immobilized primer;

(c) providing a solution which contains a DNA polymerase and only one type of dye-labeled dNTP, where N is A, T or U, G or C, or an RNA polymerase and only one type of dye-labeled NTP, where N is A, U, G or C, to said immobilized primer, and allowing the dye-labeled dNTP or NTP to react with the 3′

end of said primer, whereby the dye-labeled dNTP or NTP, which forms a base-pair with a base in the nucleic acid molecule at a position where the dye-labeled dNTP or NTP reacts with the 3′

end of said primer and is bound to the primer by action of the polymerase;

(d) detecting a bound, dye-labeled dNTP or NTP;

(e) disrupting the dye molecule of the bound, dye-labeled dNTP or NTP;

(f) repeating (c) to (e) while changing the type of dye-labeled dNTP or NTP in turn, to sequentially bind dNTPs or NTPs which forms a base-pair with the nucleotides of the nucleic acid molecule; and

(g) determining a nucleotide sequence of the nucleic acid molecule based on the types of the sequentially bound dNTPs or NTPs.

13. The method of claim 12, wherein said surface of a solid is the inner wall of a capillary.

14. The method of claim 12, wherein (d) comprises optically detecting the dye molecule of said dye-labeled dNTP or NTP.

15. The method of claim 12, wherein (d) comprises exciting dye molecules by irradiation of a laser beam and detecting a fluorescent signal.

16. The method of claim 12, wherein said detection is performed using a confocal fluorescence microscope system.

17. The method of claim 15, wherein said disrupting the dye molecules in (e)-comprises irradiating with a laser beam stronger than the laser beam in (d).

18. The method of claim 12, wherein said dye is a fluorescent dye.

19. The method of claim 12, wherein said dye-labeled dNTP is labeled with rhodamine, tetramethyl rhodamine (fluorescein) Rhodamine 6G, fluorescein isothiocyanate, or 4-fluoro-7-nitro-benzofurazon (Texas Red).

20. The method of claim 12, wherein said dye-labeled NTP is labeled with rhodamine, tetramethyl rhodamine (fluorescein) Rhodamine 6G, fluorescein isothiocyanate, or 4-fluoro-7-nitro-benzofurazon (Texas Red).

21. The method of claim 12, wherein said dNTP or NTP is each labeied with the same dye.