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    • 5. 发明申请
    • HIGHLY SENSITIVE METHOD FOR DETECTING LOW FREQUENCY MUTATIONS
    • 用于检测低频突变的高灵敏度方法
    • WO2013123031A3
    • 2015-03-12
    • PCT/US2013025913
    • 2013-02-13
    • JANSSEN DIAGNOSTICS LLC
    • WANG HAIYINGJIANG YUQIUWANG YIXIN
    • C07H21/04C12Q1/68
    • C12Q1/6827C12Q1/6858C12Q2525/186C12Q2537/163
    • The disclosed edge-blocker oligonucleotide based AS-NEPB-PCR method amplifies allele specific DNA (or RNA) while dramatically blocking amplification of wild type (WT) DNA (or RNA). The AS-NEPB-PCR design allows ready modification of an existing PCR reaction setup with an edge-blocker oligonucleotide together with an allele specific primer complementary to the mutant sequence to achieve allele specific amplification. The method simplifies assay optimization procedures and achieved sensitivity sufficient to detect a signal present at 0.1% level with close to 100% specificity, which is useful in detecting SNP or genetic mutations. The method was used to detect three different genetic mutations in cancer, in KRAS, BRAF, and EGFR, with three different types of modified edge-blocker oligonucleotides (phosphate, inverted dT and amino-C7). It was possible to detect one copy of mutant DNA in 1000-copy of normal DNA background of a heterogeneous sample, and was far more sensitive than the other blocking method.
    • 所公开的基于边缘阻断剂寡核苷酸的AS-NEPB-PCR方法扩增等位基因特异性DNA(或RNA),同时显着阻断野生型(WT)DNA(或RNA)的扩增。 AS-NEPB-PCR设计允许利用边界阻断剂寡核苷酸与等位基因特异性引物与突变序列互补的等位基因特异性引物即可修改现有PCR反应设置,以实现等位基因特异性扩增。 该方法简化了测定优化程序,并实现了足以检测0.1%水平的信号,具有接近100%的特异性的灵敏度,可用于检测SNP或遗传突变。 该方法用于检测癌症,KRAS,BRAF和EGFR中的三种不同基因突变,其中三种不同类型的修饰的边缘阻断剂寡核苷酸(磷酸盐,反向dT和氨基-C C7)。 可以在异质样品的正常DNA背景的1000拷贝中检测到一个拷贝的突变体DNA,并且比其他阻断方法更敏感。