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

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...

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 needed; the combined method does not have any risk of contamination with amplified products; (3) the assay is hard to be affected by inhibitors because the combined method is a competitive method; (4) since a gene-amplifying processing-step and a detecting processing step of a amplified product can be completely divided, a large quantity-sample becomes to be treated; and a sample-treating power can be conveniently and non-expensively improved (for example, subsequent to the gene-amplification by using plural non-expensive PCR-apparatuses not having any fluorescence-measuring function, by analyzing obtained data in order using a fluorescence-measuring apparatus, using even one fluorescence-measuring apparatus, a large quantitative-sample can be treated.); (5) a nucleic acid becomes to be assayed by a markedly simple and non-expensive measuring apparatus because the amplifying processing of a nucleic acid is needed not to be monitored in a real time way and the measuring apparatus is needed to have a thermally-cycling function indispensable to measuring a PCR processing.

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 higher in concentrations, a Tm value is higher; if contrary to the former, it becomes lower); as a result, there is such problems that (1) an assaying system needs to be optimized in every concentration of a probe, and (2) a calculating curve needs to be prepared in every concentration of an added probe.

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