Novel mixtures for assaying nucleic acid, novel method of assaying nucleic acid with the use of the same and nucleic acid probe to be used therefor

Inactive Publication Date: 2007-06-07
KANKYO ENJINIARINGU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0109] A novel method having the following characteristics is completed by using the above novel mixture according to the present invention. That is, in the method for assaying a nucleic acid, 1) processing steps for diluting a target nucleic acid are not required; 2) a change of the concentration of a nucleic acid for use with response to the concentration of a target nucleic acid are not required; 3) plural target nucleic acids can be assayed in a like assaying system; 4) an assaying sensitivity is enhanced; and 5) the combination of a method for assaying a nucleic acid according to the present invention and a method for amplifying a nucleic acid provides the following merits:
[0110] (1) subsequent to the completion of a gene amplifying reaction, the combined method makes it possible to assay in a rapid and convenient way a nucleic acid without opening a reaction tube for the gene amplifying reaction; therefore, it does not require a PCR post processing step and can simply, easily and rapidly assay a nucleic acid; (2) to open a gene-amplifying reaction tube is not n

Problems solved by technology

The above 1) requires a diluting processing step; the operation become complicated.
As results, the above 2) has such problems that (1) long assaying time is needed; (2) diluting errors occur; and (3) on automation of the assaying, an for dilution is needed.
If a target nucleic acid and a probe are hig

Method used

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  • Novel mixtures for assaying nucleic acid, novel method of assaying nucleic acid with the use of the same and nucleic acid probe to be used therefor
  • Novel mixtures for assaying nucleic acid, novel method of assaying nucleic acid with the use of the same and nucleic acid probe to be used therefor
  • Novel mixtures for assaying nucleic acid, novel method of assaying nucleic acid with the use of the same and nucleic acid probe to be used therefor

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0424] A novel mixture comprising two QProbes and a novel method for assaying a gene making use thereof.

[0425] Novel Mixture

[0426] The novel mixture having the following composition was prepared. [0427] The following QProbe A (a QProbe for detecting a target gene): 200 nM; [0428] the following QProbe B (for detecting a internal standard gene): 200 nM; [0429] the following internal standard nucleic acid: the ones [0430] having each of the following concentrations were prepared:

a) 1 / 10, 1 / 5, 1 / 2, 4 / 5, and 9 / 10 of 200 nM

b) 1 / 10, 1 / 5, 1 / 2, 4 / 5, and 9 / 10 of 800 nM; [0431] Buffer composition: 10 mM Tris-HCl buffer (pH: 8.3), KCl: 50 mM, MgCl2: 1.5 mM

[0432] Novel Assaying Method

[0433] (1) Experimental Method

[0434] It is examined whether or not a ratio between genes existing a system is capable of being determined using the above mixture added with a target gene and using two QProbes. In the experiment, after DNA solutions with varied ratios between a target gene and an internal st...

example 2

[0456] 2-2 Screening of Novel Dye Capable of Quenching Fluorescence Due to its Co-Reaction with a Guanine.

[0457] (1) Experimental Method

[0458] A probe was prepared in which said probe was labeled at a C of a sugar of the 3′-end with a dye to be screened. A fluorescence-quenching rate was assayed by making the prepared probe hybridize with a corresponding chain in a solution.

[0459] Experimental Procedures

[0460] (2) Experimental Conditions

[0461] Target Gene

[0462] Target gene: an oligo DNA was used.

[0463] Synthesis of oligonucleotide: by relying custom synthesis services (Espec oligoservices Inc.).

Base sequence of target3′ggggg ggggg ggAAA AAA5′gene:

[0464] Base sequence of internal standard gene:

[0465] Final concentration: 320 nM.

[0466] Probe

[0467] Synthesis of oligonucleotide: by relying custom synthesis services (Espec oligoservices Inc.).

Base sequence:5′CCCCC CCCCC CCTTT TTT3′

Dye: PacificBlue (P-10163), TET (C-6166), TBSF (C-6166), HEX (T-20091), and R6G (C-6127) were...

example 3

[0476] 2-3 Method for Assaying a Gene Using a Switching Probe Novel Mixture

[0477] The novel mixture having the following composition was prepared. [0478] The below Switching probe: 400 nM; [0479] the following internal standard nucleic acid: the ones [0480] having the following concentrations each were prepared: (a) 1 / 10, 1 / 5, 1 / 2, 4 / 5, and 9 / 10 of 600 nM [0481] Buffer composition: 10 mM Tris-HCl buffer (pH: 8.3), KCl: 50 mM, MgCl2: 1.5 mM.

[0482] In addition, in order to verify the experimental data obtained by using the Switching probe, a novel mixture was prepared by replacing the following QProbe A and QProbe B for the Switching probe in the above reaction solution—The following QProbe A (for detecting a target gene): 200 nM; [0483] The following QProbe B (for detecting an internal standard gene): 200 nM.

[0484] Novel Assaying Method

[0485] A target gene was assayed by adding the target gene in the above novel mixture. Further, the fluorescent quenching rates obtained by using ...

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Abstract

[Problems] To provide a novel mixture for assaying a target nucleic acid, characterized by enabling a nucleic acid assay while: 1) requiring no step of diluting the target nucleic acid; 2) requiring no procedure of changing a probe concentration depending on a concentration of the target nucleic acid. [Means for Solving Problems] 1) A mixture which comprises one internal standard nucleic acid and two nucleic acid probes labeled with a fluorescent dye; 2) a mixture for measuring Km value which comprises one internal standard nucleic acid having a partial mutation and one nucleic acid probe labeled with a fluorescent dye; 3) a mixture which comprises one internal standard nucleic acid and one double nucleic acid probe labeled with two fluorescent dyes; and a method for assaying a nucleic acid by making use thereof.

Description

TECHNICAL FIELD [0001] This invention relates to a novel mixture for assaying a nucleic acid, and specifically to a novel mixture for assaying accurately, conveniently and non-expensively one or plural nucleic acids, and a method for assaying a nucleic acid using the same, and nucleic acid probes to be used for the assaying method and a nucleic acid-assaying kit. BACKGROUND ART [0002] Many examples of methods for assaying a target nucleic acid are methods for assaying a nucleic acid in a homogenous solution by using a fluorescent dye-labeled target nucleic acid probe having properties changing in fluorescence on hybridizing with a target nucleic acid (called a nucleic acid probe for a homogenous solution system or a target nucleic acid probe, having the same meaning). The method does not require, by this characteristics, 1) any step immobilizing a target nucleic acid (or any step washing a probe) indispensable for common hybridizing method and 2) any step washing out non-reacting pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C07H21/04G01N21/64G01N33/53G01N33/58
CPCC12Q1/6818Y10T436/143333C12Q2545/107
Inventor NAKAMURA, KAZUNORIKANAGAWA, TAKAHIRONODA, NAOHIROTSUNEDA, SATOSHITANI, HIDENORIKURATA, SHINYA
Owner KANKYO ENJINIARINGU
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