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1.发明公开Sequence-specific engineered ribonuclease H and the method for determining the sequence preference of DNA-RNA hybrid binding proteins 审中-公开序列特异性修饰核糖核酸酶H和方法，用于确定DNA-RNA杂交序列优先结合蛋白质的
公开(公告)号：EP2857506A3
公开(公告)日：2015-04-29
申请号：EP14180902.0
申请日：2012-06-07
IPC分类号： C12N9/22
CPC分类号： C12N9/22 , C07K14/435 , C12Y301/26004
摘要： The present invention relates to the of determining the sequence preference of DNA-RNA hybrid binding protein or its domain which comprises the following steps:
a) contacting of the purified protein or its domain, with a mixture of a library of DNA-RNA hybrids substrates, wherein DNA-RNA hybrid substrates comprise randomized sequences in the central part, preferably randomized in 9 or 10 nucleotide positions, with flanking sequences fixed and allowing a tested protein or its domain to bind the sequence to which it has an affinity;
b) separating unbound DNA-RNA hybrids by immobilization of protein or its domain, with bound DNA-RNA hybrid, to the resin, preferably glutathione agarose;
c) removing the unbound hybrids;
d) isolating the recombinant protein, together with the associated DNA-RNA hybrids,
e) amplification of the isolated hybrid using PCR, preferably RT-PCR, wherein in the amplification reaction the primers complementary to invariant sequences flanking the randomized region are used, and wherein during the PCR reaction on one of the primer the sequence of RNA polymerase promoter is added in order to obtain double-stranded DNA for in vitro transcription with RNA polymerase;
f) reverse transcription using reverse transcriptase that does not possess the RNase H activity and a DNA primer complementary to the 3' end of the RNA template in order to obtain a DNA-RNA hybrid..-
2.发明公开Sequence-specific engineered ribonuclease H and the method for determining the sequence preference of DNA-RNA hybrid binding proteins 审中-公开序列特异性修饰核糖核酸酶H和方法，用于确定DNA-RNA杂交序列优先结合蛋白质的
公开(公告)号：EP2857506A2
公开(公告)日：2015-04-08
申请号：EP14180902.0
申请日：2012-06-07
IPC分类号： C12N9/22
CPC分类号： C12N9/22 , C07K14/435 , C12Y301/26004
摘要： The present invention relates to the of determining the sequence preference of DNA-RNA hybrid binding protein or its domain which comprises the following steps:
a) contacting of the purified protein or its domain, with a mixture of a library of DNA-RNA hybrids substrates, wherein DNA-RNA hybrid substrates comprise randomized sequences in the central part, preferably randomized in 9 or 10 nucleotide positions, with flanking sequences fixed and allowing a tested protein or its domain to bind the sequence to which it has an affinity;
b) separating unbound DNA-RNA hybrids by immobilization of protein or its domain, with bound DNA-RNA hybrid, to the resin, preferably glutathione agarose;
c) removing the unbound hybrids;
d) isolating the recombinant protein, together with the associated DNA-RNA hybrids,
e) amplification of the isolated hybrid using PCR, preferably RT-PCR, wherein in the amplification reaction the primers complementary to invariant sequences flanking the randomized region are used, and wherein during the PCR reaction on one of the primer the sequence of RNA polymerase promoter is added in order to obtain double-stranded DNA for in vitro transcription with RNA polymerase;
f) reverse transcription using reverse transcriptase that does not possess the RNase H activity and a DNA primer complementary to the 3' end of the RNA template in order to obtain a DNA-RNA hybrid..摘要翻译： 本发明涉及DNA-RNA杂交物结合蛋白或其结构域的确定性挖掘的序列偏好其包括以下步骤：a）使纯化的蛋白质或其结构域，具有DNA-RNA杂交基板的文库的混合物 ，worin DNA-RNA杂交基板包含随机序列在中心部分，优选地随机分为9个或10个核苷酸位置，与侧翼序列和固定的允许一个测试蛋白质或其结构域的序列结合到由它来亲和力; b）在蛋白质或其结构域的固定化分离未结合的DNA-RNA杂交体，具有结合的DNA-RNA杂交，以该树脂，优选谷胱甘肽琼脂糖; c）除去未结合的杂种; d）在使用PCR，优选RT-PCR，worin在扩增反应中的引物与侧翼于随机化区域不变序列互补的分离的杂交体的相关的DNA-RNA杂交体，E）扩增分离重组蛋白质，一起被使用，并且 上的RNA聚合酶启动子的序列，以获得用于在体外与RNA聚合酶转录的双链DNA中加入引物中的一个PCR反应期间worin; F）使用逆转录酶没有不具有RNA酶H活性和DNA引物，以便获得一个DNA-RNA杂交到模板RNA的3“末端互补的反转录..
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