METHOD FOR AMPLIFICATION OF TARGET NUCLEIC ACID SEQUENCE, METHOD FOR DETECTION OF MUTATION BY USING THE METHOD, AND REAGENTS FOR USE IN THE METHODS

摘要

Disclosed is a mutation detection method which can detect a mutation in a single reaction system with excellent sensitivity and reliability. Primers (Xmt) and (Xwt) are used to amplify a target nucleic acid sequence in which a nucleotide to be detected is mutated with higher amplification efficiency than that for a target nucleic acid sequence in which the nucleotide to be detected is normal. The primer (Xmt) is complementary to a region containing a mutated nucleotide in a template nucleic acid and has a nucleotide complementary to the mutated nucleotide in the 3' region thereof. The primer (Xwt) is complementary to a region containing the normal nucleotide in a template nucleic acid and has a nucleotide complementary to the normal nucleotide in the 3' region thereof. It is preferred that the efficiency of the amplification of the mutated template nucleic acid using the primer (Xmt) is higher than that of the amplification of the normal template nucleic acid using the primer (Xwt). Subsequently, a signal value which reflects the melting state of a hybrid of the amplification product obtained in the preceding step and the probe is measured. The presence or absence of the mutation of the nucleotide of interest is determined based on the variation in the signal value in accordance with the change in temperature.