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1. 发明申请

WO2015121675A1 METHYLATION DETECTION METHOD 审中-公开
标题翻译：甲基化检测方法
公开(公告)号：WO2015121675A1
公开(公告)日：2015-08-20
申请号：PCT/GB2015/050422
申请日：2015-02-13
申请人： BASE4 INNOVATION LTD
发明人： BALMFORTH, Barnaby , SILVA, Ana Luisa Bras dos Santos Ribeiro da
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6827 , C12Q2521/319 , C12Q2521/331 , C12Q2521/501 , C12Q2525/125 , C12Q2525/301 , C12Q2565/107
摘要： A method of determining whether a given single nucleotide is methylated or not methylated characterised by the steps of (a) contacting the single nucleotide with one or more hybridisation probe types each of which in its unused form comprises (1) a first oligonucleotide to which is attached one or more first detectable elements in an essentially undetectable state and which further comprises (i) double- and single-stranded regions and (ii) a region resistant to exonucleolytic degradation attached to the end of the double-stranded region adjacent the single- stranded region and (2) a second single-stranded oligonucleotide to which is attached one or more second detectable elements also in an essentially undetectable state and which is adapted to be at least in part the nucleotide sequence compliment of the single-stranded region of the first oligonucleotide; (b) for the relevant probe type causing (i) the single nucleotide to bind to the region resistant to exonucleolytic degradation and the single-stranded region and (ii) the second oligonucleotide to bind to the single nucleotide and the single-stranded nucleotide region to create a substantially double-stranded used probe; either (c1) treating the used probe with a methylation-dependent restriction endonuclease under conditions whereby the used probe is cleaved adjacent the region resistant to exonucleolytic degradation into two double-stranded oligonucleotide products if the single nucleotide is methylated or (c2) treating the used probe with a methylation-sensitive restriction endonuclease under conditions whereby the used probe is cleaved adjacent the region resistant to exonucleolytic degradation into two double-stranded oligonucleotide products if the single nucleotide is not methylated; and thereafter either (d1) treating the product of step (c1) with an exonuclease or a polymerase exhibiting exonuclease activity to liberate either only first or both first and second detectable elements in a detectable state if the single nucleotide is methylated, or only the second detectable elements if not or (d2) treating the product of step (c2) with an exonuclease or a polymerase exhibiting exonuclease activity to liberate either only first or both first and second detectable elements in a detectable state if the single nucleotide is not methylated, or only the second detectable element if the single nucleotide is methylated.
摘要翻译：确定给定的单核苷酸是否被甲基化或未甲基化的方法，其特征在于以下步骤：（a）使单个核苷酸与一种或多种其未使用形式的杂交探针类型接触，所述杂交探针类型包含（1）第一寡核苷酸连接一个或多个第一可检测元件，其基本上不可检测的状态，并且还包含（i）双链和单链区域和（ii）与邻近单链区域的双链区域末端附着的外源核酸水解降解的区域，双链区域和（2）第二单链寡核苷酸，其连接有一个或多个第二可检测元件，其也处于基本上不可检测的状态，并且其适于至少部分地基于所述单链区域的核苷酸序列互补第一寡核苷酸; （b）对于相关探针类型，导致（i）单核苷酸结合抗外切核酸分解降解区域和单链区域，和（ii）第二寡核苷酸结合单核苷酸和单链核苷酸区域以产生基本上双链的探针; （c1）在甲基化依赖性限制性内切核酸酶处理所使用的探针的条件下，如果单核苷酸被甲基化，或（c2）处理所使用的（c2）处理所用的探针在邻近外切核酸分解降解的区域裂解成两个双链寡核苷酸产物在甲基化敏感性限制性内切核酸酶的探针条件下，如果单个核苷酸未被甲基化，则使用的探针在抗核外裂解降解的区域附近裂解成两条双链寡核苷酸产物; 然后（d1）用外切核酸酶或表达核酸外切酶活性的聚合酶处理步骤（c1）的产物，以仅在第一个或第二个可检测元件处于可检测状态时释放，如果单个核苷酸被甲基化，或只有第二个如果不是，或（d2）用外切核酸酶或表达核酸外切酶活性的聚合酶处理步骤（c2）的产物，如果单个核苷酸没有被甲基化，则只有第一个或第二个可检测元件在可检测的状态下释放，或者只有第二个可检测元素，如果单个核苷酸是甲基化的。

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