Method of assaying nucleic acid using labeled nucleotide

• US 7,785,795 B2
• Filed: 04/10/2009
• Issued: 08/31/2010
• Est. Priority Date: 05/31/2002
• Status: Expired due to Fees

• Current Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan), Kankyou Engineering Co., Ltd.
• Original Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan), Kankyou Engineering Co., Ltd.
• Inventors: Kanagawa, Takahiro, Takatsu, Kyoko, Nakamura, Kazunori, Kurata, Shinya
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US12/421,983
• Publication Number:
  US 20090233308A1
• Time in Patent Office: 508 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/6.11
• 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 quantifying at least one nucleic acid, which comprises:

conducting a nucleic acid polymerization reaction in a nucleic acid polymerization reaction system that is a reaction solution comprising;

(A) said at least one nucleic acid as a template,

(B)(a) a fluorescent dye capable of emitting fluorescence upon binding to a nucleic acid; and

(b) a nucleotide monomer labeled with an acceptor fluorescent dye, and

(C) at least one nucleic acid-synthesizing enzyme,

measuring in real-time during the nucleic acid polymerization a change or an amount of a change in an optical character of said nucleic acid polymerization system as the reaction solution; and

determining the quantity of said template nucleic acid or nucleic acid, being synthesized using said template nucleic acid as a template based on the measured change or the measured amount of the change in the optical character.

2. The method according to claim 1, wherein said nucleic acid polymerization system further comprises (D) a nucleic acid primer capable of specifically hybridizing to said template nucleic acid.

3. The method according to claim 2, wherein said nucleic acid primer (D) is a single unlabeled nucleic acid primer, which is selected from the group consisting of a forward primer, a reverse primer, and a primer that is both a forward and reverse primer.

4. The method according to claim 1, wherein said fluorescent dye (B)(a) is an intercalator.

5. The method according to claim 1, wherein said optical character comprises an optical character derived from said fluorescent dye (B)(a) and said acceptor fluorescent dye, with which said nucleotide monomer (B)(b) is labeled.

6. The method according to claim 1, wherein said nucleotide monomer (B)(b) comprises at least two kinds of nucleotide monomers which differ in base from each other, wherein said two kinds of nucleotide monomers are labeled with different acceptor fluorescent dyes, respectively, wherein said optical character is derived from said acceptor fluorescent dyes.

7. A method of detecting a single nucleotide polymorphism in a nucleic acid template, the method comprising:

conducting a single-base elongation reaction in a nucleic acid polymerization reaction system that is a reaction solution, wherein the system comprises

(A) the nucleic acid as a template,

(B) (a) a fluorescent dye capable of emitting fluorescence upon binding to a nucleic acid; and

(b) a dideoxynucleotide monomer labeled with an acceptor fluorescent dye,

(C) at least one nucleic acid-synthesizing enzyme, and

(D) a nucleic acid primer,

measuring in real-time during the single-base elongation reaction a change or an amount of change in an optical character of the nucleic acid polymerization reaction system as the reaction solution; and

detecting the single nucleotide polymorphism based on the measured change or the measured amount of the change in the optical character.

8. The method according to claim 7, wherein said nucleic acid primer (D) is designed such that the 3′

end of the primer is complementary to a single nucleotide polymorphism site of the nucleic acid template.

9. The method according to claim 7, wherein said nucleic acid primer (D) is a single unlabeled nucleic acid primer, which is selected from the group consisting of a forward primer, a reverse primer, and a primer that is both a forward and reverse primer.

10. The method according to claim 7, wherein said fluorescent dye (B)(a) is an intercalator.

11. The method according to claim 7, wherein said optical character comprises an optical character derived from said fluorescent dye (B)(a) and said acceptor fluorescent dye, with which said dideoxynucleotide monomer (B)(b) is labeled.

12. The method according to claim 7, wherein said dideoxynucleotide monomer (B)(b) comprises at least two kinds of dideoxynucleotide monomers which differ in base from each other, wherein said two kinds of dideoxynucleotide monomers are labeled with different acceptor fluorescent dyes, respectively, wherein said optical character is derived from said acceptor fluorescent dyes.