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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 thereforRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidNovel 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 description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070128608, 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. Brief Patent Description - Full Patent Description - Patent Application Claims
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 probes (or any step immobilizing non-reacting genes); the method, therefore, is a method for assaying easily, rapidly and accurately a target nucleic acid. With such reasons, the method for assaying quantitatively a target nucleic acid has been in a wide used for various methods for analyzing genes (see non-patent reference 1). [0003] In assaying directly a target nucleic acid by a nucleic acid probe for a homogenous solution system, an object could be achieved by using the following procedures. (1) making a target nucleic acid of known concentrations ready beforehand hybridize with a nucleic acid probe for a homogenous solution system, and, on this hybridization, monitoring a change or the amount of change in an optical character; [0004] (2) preparing a calculating curve for determining a target nucleic acid by preparing an equation relating to the above change or amount of change in an optical character and amounts of a target nucleic acid because the change or the amount of change is positively proportional to amounts of a target nucleic acid; [0005] (3) conducting a procedures similar to the above procedures in regard to an unknown sample, and determining the amount of a target nucleic acid from the above calculating curve based on the obtained change or amount of change in an optical character. [0006] In the method, however, if a target nucleic acid exists greater in concentration than an added nucleic acid probe, a change or the amount of a change in an optical character is at any time stationary. Because of this, the conventional methods need any of the means, 1) diluting a target nucleic acid sample, and 2) preparing in advance plural assaying systems with a probe having various concentrations. 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. In the above 2) also, the reaction time and reaction temperature suitable for hybridization vary with response to the concentrations of an added probe (if a target nucleic acid and a target nucleic acid probe are higher in concentrations, the time for completed hybridization becomes shorter; if contrary to the former, the reaction time becomes longer. 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. [0007] Non-patent reference 1: Protein, Nucleic Acid and Enzyme; vol. 35, No. 17, Kyoritu Shuppan Co., Ltd.; Experimental Medical; vol. 15, No. 7, 1997, Yodosha Co., Ltd. DISCLOSURE OF THE INVENTION [0008] Problems to be Solved by Invention [0009] With the foregoing circumstances in view, the present invention has as an object to provide a novel mixture for assaying a target nucleic acid, characterized by enabling a nucleic acid assaying method to assay a target nucleic acid without requiring any processing step of diluting of a target nucleic acid and any procedures of changing a probe concentration, a novel method for assaying a nucleic acid using the same, and a novel nucleic acid probe to be used for these. MEANS TO BE SOLVED PROBLEMS [0010] As a result of an extensive investigation, the present inventors have obtained the following findings and completed the present invention. That is, a target nucleic acid is added to a reaction solution (a hybridizing reaction system) comprising a known concentration of a specific internal standard nucleic acid, a specific target nucleic acid probe labeled with a fluorescent dye for a homogenous solution system and/or a specific internal standard nucleic acid probe labeled with a fluorescent dye and making it possible to hybridize the above internal standard nucleic acid, and then, is allowed to make a hybridizing reaction; while an occurred change of an optical character is measured. Further a measured-value ratio of the internal standard nucleic acid and the target nucleic acid is determined; these steps enabled the present invention to be completed. [0011] Described specifically, the present invention provides: [0012] 1) Novel mixture or novel reaction solution (hereinafter, the both together are collectively called simply a "novel mixture".) for assaying one or two or more target nucleic acids, which comprises one or two or more below nucleic acid probes for a homogenous solution system and one or two or more below internal standard nucleic acid, or further one or two or more below internal standard nucleic acid probe: [0013] A) Nucleic acid probe for homogenous solution system (hereinafter, called a "target nucleic acid probe") having below characteristics: a) said target nucleic acid probe is formed of one stranded oligonucleotide; [0014] b) said target nucleic acid probe is labeled with one or two or more molecule of fluorescent dyes of one or two or more kinds at least one of both end portions and/or at least one of base portions in the chain, at least one of sugar moieties and/or at least one of phosphate moieties of the oligonucleotide; c) said target nucleic acid probe enables a fluorescent character to change on hybridizing with a target nucleic acid and/or an internal standard nucleic acid; d) said target nucleic acid probe is capable of hybridizing without discriminating with a target nucleic acid or an internal standard nucleic acid; e) said target nucleic acid probe is capable of producing a difference between the amount of a change in a fluorescent character on hybridizing with an internal standard nucleic acid and that on hybridizing with a target nucleic acid; [0015] B) Internal standard nucleic acid (called an "internal gene" or an "internal standard gene" in a case): wherein said internal standard nucleic acid has a structure different in at least a portion from the structure of a target nucleic acid of a region corresponding to the above target nucleic acid probe, and is capable of producing a difference between the amount of a change in a fluorescent character produced on hybridizing with the above target nucleic acid probe and one on the hybridization of a target nucleic acid with the above target nucleic acid probe; Continue reading about 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... 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