Patent ID: 9587271
Date: 2017-03-07
CPC Classifications: C12Q,Y10T

Claim:
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.