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    • 9. 发明申请
    • Covalent joining of DNA strands to RNA strands catalyzed by vaccinia topoisomerase
    • 将DNA链与牛痘拓扑异构酶催化的RNA链共价连接
    • US20100317064A1
    • 2010-12-16
    • US12658764
    • 2010-02-12
    • Stewart ShumanJoAnn SekiguchiJohn ComiskeyJoseph FernandezJames HoefflerRobert Marcil
    • Stewart ShumanJoAnn SekiguchiJohn ComiskeyJoseph FernandezJames HoefflerRobert Marcil
    • C12P19/34C12N15/64C07H21/04C12N15/63
    • C12N15/1096C07H21/02C07H21/04C12N9/90C12N15/1006C12Y599/01002
    • The present invention provides a method of covalently joining a DNA strand to an RNA strand comprising (a) forming a topoisomerase-DNA intermediate by incubating a DNA cleavage substrate comprising a topoisomerase cleavage site with a topoisomerase specific for that site, wherein the topoisomerase-DNA intermediate has one or more 5′ single-strand tails; and (b) adding to the topoisomerase-DNA intermediate an acceptor RNA strand complementary to the 5′ single-strand tail under conditions permitting a ligation of the covalently bound DNA strand of the topoisomerase-DNA intermediate to the RNA acceptor strand and dissociation of the topoisomerase, thereby covalently joining the DNA strand to the RNA strand. The present invention also provides a method of tagging a 5′ end of an RNA molecule. The present invention further provides a DNA-RNA molecule which has been joined in vitro by the use of a topoisomerase. The present invention also provides a method of tagging a 5′ end of an mRNA. The present invention provides a method of isolating and cloning full-length gene sequences using capped mRNA after subtraction of non-capped RNA.
    • 本发明提供了将DNA链与RNA链共价连接的方法,其包括(a)通过将包含拓扑异构酶切割位点的DNA切割底物与该位点特异性的拓扑异构酶一起孵育而形成拓扑异构酶-DNA中间体,其中拓扑异构酶-DNA 中间体具有一个或多个5'单链尾部; 和(b)在允许拓扑异构酶-DNA中间体与RNA受体链的共价结合的DNA链连接的条件下,向拓扑异构酶-DNA中间体添加与5'单链尾部互补的受体RNA链,并解离 从而将DNA链共价连接到RNA链上。 本发明还提供了标记RNA分子的5'末端的方法。 本发明还提供了通过使用拓扑异构酶体外结合的DNA-RNA分子。 本发明还提供了标记mRNA的5'末端的方法。 本发明提供了在减去未加帽的RNA之后使用加帽的mRNA分离和克隆全长基因序列的方法。