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    • 4. 发明公开
    • NUCLEIC ACID AMPLIFICATION METHOD USING ALLELE-SPECIFIC REACTIVE PRIMER
    • VERFAHREN ZURNUKLEINSÄUREAMPLIFIKATIONMITTELS EINES ALLELSPEZIFISCHEN REAKTIVEN PRIMERS
    • EP2982762A1
    • 2016-02-10
    • EP13881318.3
    • 2013-04-16
    • Genomictree, Inc.
    • AN, Sung WhanOH, Tae Jeong
    • C12Q1/68C12N15/11
    • C12Q1/6858C12Q2521/101C12Q2523/125
    • The present invention relates to a nucleic acid amplification method using an allele-specific reactive primer (ASRP) which is designed to solve problems of conventional allele-specific PCR. More specifically, the present invention relates to a detection method of a target nucleic acid, comprising a DNA polymerase having proofreading activity and a base sequence complementary to the target nucleic acid, wherein the target nucleic acid is amplified in the presence of an ASRP which is modified in such a manner that one or more bases from the base immediately in front, in the 5'-end direction, of a base that is cut off by the proofreading activity of the DNA polymerase when a non-complementary base exists at the 3' end, to the base at the 5' end, cannot serve as a primer for polymerization. The detection method using an ASRP according to the present invention is a technique with very high specificity of amplification due to the characteristics of the ASRP and the proofreading DNA polymerase, and is capable of effectively detecting mutations (point mutation, insertion, and deletion) including single nucleotide polymorphism (SNP). Furthermore, the method can also be used for detecting the presence of CpG methylation after bisulfite treatment, or for amplifying and detecting a target DNA starting with a desired base sequence from a DNA library.
    • 本发明涉及使用等位基因特异性反应性引物(ASRP)的核酸扩增方法,其被设计用于解决常规等位基因特异性PCR的问题。 更具体地,本发明涉及靶核酸的检测方法,其包含具有校正活性的DNA聚合酶和与靶核酸互补的碱基序列,其中靶核酸在ASRP存在下扩增,所述ASRP是 以这样的方式进行修饰,即当碱基不存在于3位时被DNA聚合酶的校正活性所切断的碱的5'端方向的一个或多个碱基 '结束,在5'端的碱基,不能作为聚合的底漆。 使用根据本发明的ASRP的检测方法是由于ASRP和校对DNA聚合酶的特征而具有非常高的扩增特异性的技术,并且能够有效地检测突变(点突变,插入和缺失),包括 单核苷酸多态性(SNP)。 此外,该方法还可以用于检测亚硫酸氢盐处理后CpG甲基化的存在,或者从DNA文库扩增和检测起始于所需碱基序列的靶DNA。