Polymorphism identification method

摘要

The present invention is to provide a method for identifying a polymorphism. The method includes performing a nucleic acid chain extension reaction and identifying the polymorphism of the nucleic acid contained in the test nucleic acid sample. The extension reaction is conducted with use of a nucleic acid in a test nucleic acid sample as a template, a type I detection primer which hybridizes with a region including the polymorphic site of a nucleic acid whose polymorphic site nucleotide sequence consisting of a first nucleotide sequence, and a polymerase. The reaction is conducted with the presence of an inhibitory oligonucleotide, which hybridizes with the type I detection primer. The region of the type I detection primer to hybridize with the inhibitory oligonucleotide is located on the 5′ side of the polymorphism detection site of the type I detection primer to hybridize with the polymorphic site.

主权项

1. A polymorphism identification method for identifying a polymorphism of a polymorphic site-containing nucleic acid, comprising:
(a) preparing a reaction mixture, the mixture comprising: a test nucleic acid sample as a template, wherein the test nucleic acid sample comprises a first nucleic acid comprising the polymorphic site; a type I detection primer, wherein the type I detection primer comprises a polymorphism detection site, the type I detection primer comprising: a template-binding region, and an additional nucleic acid sequence added to the 5′ end of the template-binding region, wherein the nucleotide sequence of the template-binding region is completely complementary to the nucleotide sequence of the first nucleic acid and not completely complementary to the nucleotide sequence of a second nucleic acid, and wherein the additional nucleic acid sequence is complementary to the nucleotide sequence adjacent to the 3′ end of the template binding region, wherein the polymorphism detection site of the type I detection primer is located within five nucleotides from the 3′ end of the type I detection primer; an inhibitory oligonucleotide being complementary to the additional nucleic acid sequence; (b) forming a complex comprising the template, the type I detection primer and the inhibitory oligonucleotide, wherein the template hybridizes the template-binding region of the type I detection primer, and wherein the inhibitory oligonucleotide hybridizes the additional nucleic acid sequence of the type I detection primer wherein a hybrid between the type I detection primer and the type I nucleic acid has a higher stability than a hybrid between the inhibitory oligonucleotide and the type I detection primer; (c) performing a nucleic acid chain extension reaction in the reaction mixture; and (d) identifying the polymorphism of the test nucleic acid sample, based on the amount of the product of the chain reaction extension reaction in step (c).