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

摘要

[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.

主权项

1. A method for assaying a target nucleic acid in a sample, which comprises:
(I) adding (i) a fluorescence-quenching probe to the sample, wherein the fluorescence-quenching probe (i) has, as one of end bases thereof, a cytosine which is labeled with a first fluorescence-quenching dye, and the fluorescence-quenching probe (i) is hybridizable with the target nucleic acid and is designed such that at least one guanine exists in the target nucleic acid at a position of from a 1st base to a 3rd base, with the proviso that a base, which is base-paired with the cytosine, is counted as the 1st base, and wherein the first fluorescence-quenching dye is selected from the group consisting of 2′,4,7,7′-tetra-chlorofluorescein, TBSF, 2′,4,4′,5′,7,7′-hexachlorofluorescein, R6G and 2-oxo-6,8-difluoro-7-hydroxy-2H-1-benzopyran-3-carboxylic acid, (II) maintaining the sample under conditions that the fluorescence-quenching probe (i) is hybridizable with the target nucleic acid, and measuring fluorescence derived from the fluorescence-quenching probe (i), (III) maintaining the sample under conditions that the fluorescence-quenching probe (i) is not hybridizable with the target nucleic acid, and measuring fluorescence derived from the fluorescence-quenching probe (i), and (IV) assaying the target nucleic acid in the sample from fluorescent intensities determined in the step (II) and step (III), wherein the fluorescence-quenching probe (i) have cytosines as both the end bases, and the end base different from the one end base is labeled with a second fluorescence-quenching dye different from the first fluorescence-quenching dye.