Abstract: An object of the present invention is to provide a novel method for designing a primer ensuring reactivity and discriminatory power in a method for detecting a single base substitution based on an ASP-PCR method and to provide a method for easily detecting multiple point mutations within overlapping amplicons, particularly, two adjacent single base substitutions. The single base substitutions can easily be detected by using a mutant primer in which the base of the third nucleotide from the 3? end corresponds to the base of a mutant nucleotide of a single base substitution contained in a nucleic acid sample, in which the base of the second nucleotide from the 3? end is not complementary to the base of the corresponding nucleotide of the nucleic acid, and in which the bases of the other nucleotides are complementary to the bases of the corresponding nucleotides of the nucleic acid.

Abstract: An object of the present invention is to provide a method for highly sensitively detecting, and a method for quantifying, a mutant gene (mutant allele) contained at a low frequency in a nucleic acid sample containing a wild-type allele. In one-reaction system in which a first-primers set designed to flank a mutation site of a gene of interest is mixed with a second-primers set including an ASP corresponding to the mutation, an amplification product can be obtained from a low-frequency mutant gene, and quantitativeness can be ensured, by including a competitive nucleic acid suppressing an amplification reaction from a wild-type allele of the gene, selecting a certain primer concentration condition, and controlling the cycle numbers of PCR reactions using the first and second-primers sets.

Abstract: An object of the present invention is to provide a method for accurately and quantitatively discriminating and detecting a wide variety of gene mutations, or particularly, single base substitutions or point mutations. In an ASP for analyzing gene mutations, or particularly, single base substitutions or point mutations, when a non-nucleotide component is added to the 5? end of at least one of the ASP and a primer paired therewith before amplification by PCR and amplification products thereof are separated by ion-exchange chromatography, even the amplification products having the same length can be separated and detected.

Abstract: An object of the present invention is to provide a method for accurately and quantitatively discriminating and detecting a wide variety of gene mutations, or particularly, single base substitutions or point mutations. In an ASP for analyzing gene mutations, or particularly, single base substitutions or point mutations, when a non-nucleotide component is added to the 5? end of at least one of the ASP and a primer paired therewith before amplification by PCR and amplification products thereof are separated by ion-exchange chromatography, even the amplification products having the same length can be separated and detected.

Abstract: An object of the present invention is to provide a novel method for designing a primer ensuring reactivity and discriminatory power in a method for detecting a single base substitution based on an ASP-PCR method and to provide a method for easily detecting multiple point mutations within overlapping amplicons, particularly, two adjacent single base substitutions. The single base substitutions can easily be detected by using a mutant primer in which the base of the third nucleotide from the 3? end corresponds to the base of a mutant nucleotide of a single base substitution contained in a nucleic acid sample, in which the base of the second nucleotide from the 3? end is not complementary to the base of the corresponding nucleotide of the nucleic acid, and in which the bases of the other nucleotides are complementary to the bases of the corresponding nucleotides of the nucleic acid.