Novel method of assaying nucleic acid using labeled nucleotide

• US 20060172293A1
• Filed: 05/30/2003
• Published: 08/03/2006
• Est. Priority Date: 05/31/2002
• Status: Abandoned Application

• Current Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan), Kankyou Engineering Co., Ltd.
• Original Assignee: Kankyou Engineering Co., Ltd., NATL Institute of Advanced Indus Science & Tech
• Inventors: Kurata, Shinya, Takatsu, Kyoto, Nakamura, Kazunori
• Application Number:
  US10/514,558
• Publication Number:
  US 20060172293A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

A novel method is provided to assay at least one nucleic acid. According to this method, a nucleic acid polymerization reaction is conducted in a nucleic acid polymerization reaction system, which contains (A) the at least one nucleic acid as a template, (B) at least one nucleotide monomer labeled with at least one label selected from the group consisting of (a) fluorescent dyes, (b) quenchers and (c) immune related substances with a fluorescent dye or quencher contained therein, and (C) at least one nucleic acid-synthesizing enzyme. The template nucleic acid or a nucleic acid, which has been synthesized using the template nucleic acid as a template, is then assayed from a change or an amount of a change in an optical character of the nucleic acid polymerization system. This method makes it possible to specifically and accurately assay at least one nucleic acid, which is contained in a single system and can be an unknown nucleic acid and/or a known nucleic acid, with excellent sensitivity, in short time and with ease.

Claims

1. A method of assaying at least one nucleic acid, which comprises:

conducting a nucleic acid polymerization reaction in a nucleic acid polymerization reaction system comprising (A) said at least one nucleic acid as a template, (B) at least one nucleotide monomer labeled with at least one label selected from the group consisting of (a) fluorescent dyes, (b) quenchers and (c) immune related substances with a fluorescent dye or quencher contained therein, and (C) at least one nucleic acid-synthesizing enzyme; and

assaying said template nucleic acid or a nucleic acid, which has been synthesized using said template nucleic acid as a template, from a change or an amount of a change in an optical character of said nucleic acid polymerization system.

2. A method according to claim 1, wherein said label is a combination of at least one donor fluorescent dye and at least one acceptor fluorescent dye.

3. A method according to claim 1, wherein said label is a combination of at least one fluorescent dye and at least one quencher.

4. A method according to claim 1, wherein said nucleic acid polymerization system further comprises (D) at least one nucleotide monomer not labeled with any label.

5. A method according to claim 1, wherein said nucleic acid polymerization system further comprises (E) a nucleic acid primer capable of specifically hybridizing to said template nucleic acid and comprising at least one nucleotide monomer.

6. A method according to claim 5, wherein said nucleic acid primer is labeled with (E′

) a label as defined in claim 1.

7. A method according to claim 1, wherein said nucleic acid polymerization system further comprises a non-labeled nucleotide.

8. A method according to claim 7, wherein said fluorescence-labeled and/or non-labeled nucleotide comprises guanine (g) and/or said template nucleic acid comprises at least one guanine (g).

9. A method according to claim 1, wherein said nucleic acid polymerization system further comprises a non-labeled nucleotide and a fluorescence-labeled nucleic acid primer without any ingredient (B) as defined in claim 1.

10. A method according to claim 9, wherein said non-labeled nucleotide comprises a guanine (g) base.

11. A method according to any one of claims 1, 7 and 9, wherein said non-labeled nucleotide and/or labeled nucleotide is a triphosphate.

15. A method according to any one of claims 1, 7, 9 and 12, wherein said nucleic acid polymerization system further comprises (F) a fluorescent dye capable of emitting fluorescence upon binding to a nucleic acid.

18. A method according to any one of claims 1, 7, 9, 12 and 16, wherein said nucleic acid-synthesizing enzyme is at least one enzyme selected from DNA polymerases, RNA polymerases, reverse transcriptases, and modifications thereof.

12. A method of assaying a nucleic acid, which comprises:

conducting a nucleic acid polymerization reaction in a nucleic acid polymerization system comprising said nucleic acid as a template, at least one dideoxynucleotide monomer labeled with at least one fluorescent dye and/or at least one quencher, and a nucleic acid-synthesizing enzyme; and

assaying said template nucleic acid or a nucleic acid polymer, which has been synthesized using said template nucleic acid as a template, from a change or an amount of a change in fluorescence intensity.

13. A method according to claim 12, wherein said nucleic acid polymerization system is a system further comprising a labeled nucleotide or a non-labeled nucleotide or both of them.

14. A method according to claim 12, wherein said nucleic acid polymerization system further comprises a non-labeled nucleic acid primer.

17. A method according to claim 12 or 16, wherein said non-labeled dideoxynucleotide, labeled dideoxynucleotide, non-labeled nucleotide and/or labeled nucleotide is a triphosphate.

16. A method of assaying a nucleic acid, which comprises:

conducting a nucleic acid polymerization reaction in a nucleic acid polymerization system comprising said nucleic acid as a template, a non-labeled dideoxynucleotide monomer, a labeled nucleotide, a non-labeled nucleic acid primer, at least one fluorescent dye cable of emitting fluorescence upon binding to a nucleic acid, and a nucleic acid-synthesizing enzyme;

and assaying said template nucleic acid or a nucleic acid polymer, which has been synthesized using said template nucleic acid as a template, from a change or an amount of a change in fluorescence intensity.