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    • 6. 发明申请
    • METHODS AND COMPOSITIONS FOR ENRICHMENT OF NUCLEIC ACIDS IN MIXTURES OF HIGHLY HOMOLOGOUS SEQUENCES
    • 用于在高度同源序列的混合物中增殖核酸的方法和组合物
    • WO2013003630A3
    • 2013-03-14
    • PCT/US2012044710
    • 2012-06-28
    • KUTYAVIN IGOR
    • KUTYAVIN IGOR
    • C12Q1/68C12N15/10C12N15/11
    • C12Q1/6848C12Q1/28C12Q1/6858C12Q2531/113C12Q2535/131C12Q2561/109
    • Provided are methods of enrichment and detection of target nucleic acids during target amplification in the presence of excess amounts of highly homologous sequences, said methods having substantial diagnostic utility (e.g., cancer diagnostics). Provided are amplification reaction mixtures having at least one cleavage-directing oligonucleotide, the respective binding sites of which, for the target and homologous sequences, include one or more nucleotide positions differing in sequence between the target homologous sequences. During target sequence amplification in the presence of DNA polymerase activity, a FEN1 activity, one or more amplification primers, deoxynucleoside 5'-triphosphates and other reagents suitable to support amplification of both target and homologous nucleic acid sequences, the cleavage-directing oligonucleotide hybridizes to the homologous sequence and its amplification products resulting in a FEN1-mediated cleavage of these sequences providing for increased amounts of the target nucleic acid sequence relative to that of the homologous nucleic acid sequences.
    • 提供了在存在过量高度同源序列的靶扩增过程中富集和检测靶核酸的方法,所述方法具有显着的诊断效用(例如癌症诊断)。 提供的是具有至少一个切割指导寡核苷酸的扩增反应混合物,其针对和同源序列的各自的结合位点包括靶序列之间序列不同的一个或多个核苷酸位置。 在存在DNA聚合酶活性的靶序列扩增中,FEN1活性,一种或多种扩增引物,脱氧核苷5'-三磷酸和适于支持靶和同源核酸序列扩增的其它试剂,切割定向寡核苷酸与 同源序列及其扩增产物导致这些序列的FEN1介导的切割,提供相对于同源核酸序列的靶核酸序列的增加的量。