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1. 发明申请

US20100317064A1 Covalent joining of DNA strands to RNA strands catalyzed by vaccinia topoisomerase 有权
标题翻译：将DNA链与牛痘拓扑异构酶催化的RNA链共价连接
公开(公告)号：US20100317064A1
公开(公告)日：2010-12-16
申请号：US12658764
申请日：2010-02-12
申请人： Stewart Shuman , JoAnn Sekiguchi , John Comiskey , Joseph Fernandez , James Hoeffler , Robert Marcil
发明人： Stewart Shuman , JoAnn Sekiguchi , John Comiskey , Joseph Fernandez , James Hoeffler , Robert Marcil
IPC分类号： C12P19/34 , C12N15/64 , C07H21/04 , C12N15/63
CPC分类号： C12N15/1096 , C07H21/02 , C07H21/04 , C12N9/90 , C12N15/1006 , C12Y599/01002
摘要： 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.
摘要翻译：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。

2. 发明授权

US08192932B2 Covalent joining of DNA strands to RNA strands catalyzed by vaccinia topoisomerase 有权
标题翻译：将DNA链与牛痘拓扑异构酶催化的RNA链共价连接
公开(公告)号：US08192932B2
公开(公告)日：2012-06-05
申请号：US12658764
申请日：2010-02-12
申请人： Stewart Shuman , JoAnn Sekiguchi , John Comiskey , Joseph Fernandez , James Hoeffler , Robert Marcil
发明人： Stewart Shuman , JoAnn Sekiguchi , John Comiskey , Joseph Fernandez , James Hoeffler , Robert Marcil
IPC分类号： C12Q1/68 , C12P21/06 , C12P19/34 , C07H21/02
CPC分类号： C12N15/1096 , C07H21/02 , C07H21/04 , C12N9/90 , C12N15/1006 , C12Y599/01002
摘要： The present invention provides a method of covalently joining a DNA strand to an RNA strand, a method of tagging a 5′ end of an RNA molecule, a DNA-RNA molecule which has been joined in vitro by the use of a topoisomerase, a method of tagging a 5′ end of an mRNA, and a method of isolating and cloning full-length gene sequences using capped mRNA after subtraction of non-capped RNA.
摘要翻译：本发明提供了将DNA链与RNA链共价连接的方法，标记RNA分子的5'端的方法，通过使用拓扑异构酶体外结合的DNA-RNA分子，标记mRNA的5'末端，以及在减去未加帽的RNA之后使用加帽的mRNA分离和克隆全长基因序列的方法。

3. 发明授权

US07662556B2 Covalent joining of DNA to RNA by vaccinia topoisomerase and uses thereof 有权
标题翻译：通过牛痘拓扑异构酶将DNA共价连接到RNA及其用途
公开(公告)号：US07662556B2
公开(公告)日：2010-02-16
申请号：US10666486
申请日：2003-09-19
申请人： Stewart Shuman , JoAnn Sekiguchi , Joseph Fernandez , Robert Marcil , James Hoeffler , John Comiskey
发明人： Stewart Shuman , JoAnn Sekiguchi , Joseph Fernandez , Robert Marcil , James Hoeffler , John Comiskey
IPC分类号： C12Q1/68 , C12P21/06 , C12P19/34
CPC分类号： C12N15/1096 , C07H21/02 , C07H21/04 , C12N9/90 , C12N15/1006 , C12Y599/01002
摘要： 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.
摘要翻译：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。

4. 发明申请

US20070128724A1 System for the rapid manipulation of nuculeic acid sequences 审中-公开
标题翻译：用于快速操作核酸序列的系统
公开(公告)号：US20070128724A1
公开(公告)日：2007-06-07
申请号：US11340846
申请日：2006-01-27
申请人： David Miles , Lyle Turner , Robert Marcil , Gina McConnell
发明人： David Miles , Lyle Turner , Robert Marcil , Gina McConnell
IPC分类号： C12N15/09
CPC分类号： C12N15/66 , C12N15/10 , C12N15/64 , C12P19/34
摘要： The present invention is a cell-free subcloning system utilizing three elements: (1) a donor vector that contains a nucleic acid sequence to be transferred to another vector flanked by a site-specific recombination sequence and one or more optional additional nucleic acid sequences, (2) an acceptor vector that contains a site-specific recombination sequence and one or more optional additional nucleic acid sequences, and (3) a site-specific recombinase that recognizes the site-specific recombination sequences in the donor and acceptor vectors so as to transfer the transfer sequence from the donor to the acceptor vector upon contact of the three elements of the system. Also disclosed are rapid subcloning methods employing the vectors and enzymes disclosed herein and kits for use in such methods.
摘要翻译：本发明是一种利用三个元件的无细胞亚克隆系统：（1）供体载体，其含有要转移到另一个载体，其侧翼为位点特异性重组序列和一个或多个任选的另外的核酸序列，（2）包含位点特异性重组序列和一个或多个任选的另外的核酸序列的受体载体，和（3）识别供体和受体载体中的位点特异性重组序列的位点特异性重组酶，以便当系统的三个元件接触时，将转移序列从供体转移到受体载体。还公开了使用本文公开的载体和酶的快速亚克隆方法以及用于这些方法的试剂盒。

5. 发明申请

US20050181417A1 System for the rapid manipulation of nucleic acid sequenaces 审中-公开
标题翻译：用于快速操作核酸序列的系统
公开(公告)号：US20050181417A1
公开(公告)日：2005-08-18
申请号：US11080810
申请日：2005-03-16
申请人： David Miles , Lyle Turner , Robert Marcil , Gina McConnell
发明人： David Miles , Lyle Turner , Robert Marcil , Gina McConnell
IPC分类号： C12N15/10 , C12N15/64 , C12P19/34 , C12Q1/68
CPC分类号： C12N15/66 , C12N15/10 , C12N15/64 , C12P19/34
摘要： The present invention is a cell-free subcloning system utilizing three elements: (1) a donor vector that contains a nucleic acid sequence to be transferred to another vector flanked by a site-specific recombination sequence and one or more optional additional nucleic acid sequences, (2) an acceptor vector that contains a site-specific recombination sequence and one or more optional additional nucleic acid sequences, and (3) a site-specific recombinase that recognizes the site-specific recombination sequences in the donor and acceptor vectors so as to transfer the transfer sequence from the donor to the acceptor vector upon contact of the three elements of the system. Also disclosed are rapid subcloning methods employing the vectors and enzymes disclosed herein and kits for use in such methods.
摘要翻译：本发明是一种利用三个元件的无细胞亚克隆系统：（1）供体载体，其含有要转移到另一个载体，其侧翼为位点特异性重组序列和一个或多个任选的另外的核酸序列，（2）包含位点特异性重组序列和一个或多个任选的另外的核酸序列的受体载体，和（3）识别供体和受体载体中的位点特异性重组序列的位点特异性重组酶，以便当系统的三个元件接触时，将转移序列从供体转移到受体载体。还公开了使用本文公开的载体和酶的快速亚克隆方法以及用于这些方法的试剂盒。

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