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公开(公告)号：US20090328244A1
公开(公告)日：2009-12-31
申请号：US12409326
申请日：2009-03-23
IPC分类号： A01K67/027 , C12P19/34 , C12N5/06 , C12N15/11
摘要： The present invention provides compositions, methods, and kits for covalently linking nucleic acid molecules. The methods include a strand invasion step, and the compositions and kits are useful for performing such methods. For example, a method of covalently linking double stranded (ds) nucleic acid molecules can include contacting a first ds nucleic acid molecule, which has a topoisomerase linked to a 3′ terminus of one end and has a single stranded 5′ overhang at the same end, with a second ds nucleic acid molecule having a blunt end, such that the 5′ overhang can hybridize to a complementary sequence of the blunt end of the second nucleic acid molecule, and the topoisomerase can covalently link the ds nucleic acid molecules. The methods are simpler and more efficient than previous methods for covalently linking nucleic acid sequences, and the compositions and kits facilitate practicing the methods, including methods of directionally linking two or more ds nucleic acid molecules.
摘要翻译： 本发明提供用于共价连接核酸分子的组合物，方法和试剂盒。 所述方法包括线入侵步骤，组合物和试剂盒对于进行这些方法是有用的。 例如，共价连接双链（ds）核酸分子的方法可以包括使第一个ds核酸分子接触，该第一个ds核酸分子具有与一个末端的3'末端连接的拓扑异构酶并且具有相同的单链（5'）突出端 末端，具有平末端的第二ds核酸分子，使得5'突出端可以与第二核酸分子的平端的互补序列杂交，并且拓扑异构酶可以共价连接ds核酸分子。 该方法比先前用于共价连接核酸序列的方法更简单和更有效，并且组合物和试剂盒有助于实施该方法，包括定向连接两个或多个ds核酸分子的方法。
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公开(公告)号：US06916632B2
公开(公告)日：2005-07-12
申请号：US09935280
申请日：2001-08-21
IPC分类号： A01K67/027 , C12N1/21 , C12N5/10 , C12N15/09 , C12N15/10 , C12N15/64 , C12N15/66 , C40B40/02 , C40B50/06 , C12P19/34 , C12N9/90 , C12N15/00 , C12P21/03
摘要： The present invention provides compositions, methods, and kits for covalently linking nucleic acid molecules. The methods include a strand invasion step, and the compositions and kits are useful for performing such methods. For example, a method of covalently linking double stranded (ds) nucleic acid molecules can include contacting a first ds nucleic acid molecule, which has a topoisomerase linked to a 3′ terminus of one end and has a single stranded 5′ overhang at the same end, with a second ds nucleic acid molecule having a blunt end, such that the 5′ overhang can hybridize to a complementary sequence of the blunt end of the second nucleic acid molecule, and the topoisomerase can covalently link the ds nucleic acid molecules. The methods are simpler and more efficient than previous methods for covalently linking nucleic acid sequences, and the compositions and kits facilitate practicing the methods, including methods of directionally linking two or more ds nucleic acid molecules.
摘要翻译： 本发明提供用于共价连接核酸分子的组合物，方法和试剂盒。 所述方法包括线入侵步骤，组合物和试剂盒对于进行这些方法是有用的。 例如，共价连接双链（ds）核酸分子的方法可以包括使第一个ds核酸分子接触，该第一个ds核酸分子具有与一个末端的3'末端连接的拓扑异构酶并且具有相同的单链（5'）突出端 末端，具有平末端的第二ds核酸分子，使得5'突出端可以与第二核酸分子的平端的互补序列杂交，并且拓扑异构酶可以共价连接ds核酸分子。 该方法比先前用于共价连接核酸序列的方法更简单和更有效，并且组合物和试剂盒有助于实施该方法，包括定向连接两个或多个ds核酸分子的方法。
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公开(公告)号：US20120129225A1
公开(公告)日：2012-05-24
申请号：US13205386
申请日：2011-08-08
摘要： The present invention provides compositions, methods, and kits for covalently linking nucleic acid molecules. The methods include a strand invasion step, and the compositions and kits are useful for performing such methods. For example, a method of covalently linking double stranded (ds) nucleic acid molecules can include contacting a first ds nucleic acid molecule, which has a topoisomerase linked to a 3′ terminus of one end and has a single stranded 5′ overhang at the same end, with a second ds nucleic acid molecule having a blunt end, such that the 5′ overhang can hybridize to a complementary sequence of the blunt end of the second nucleic acid molecule, and the topoisomerase can covalently link the ds nucleic acid molecules. The methods are simpler and more efficient than previous methods for covalently linking nucleic acid sequences.
摘要翻译： 本发明提供用于共价连接核酸分子的组合物，方法和试剂盒。 所述方法包括线入侵步骤，组合物和试剂盒对于进行这些方法是有用的。 例如，共价连接双链（ds）核酸分子的方法可以包括使第一个ds核酸分子接触，该第一个ds核酸分子具有与一个末端的3'末端连接的拓扑异构酶并且具有相同的单链（5'）突出端 末端，具有平末端的第二ds核酸分子，使得5'突出端可以与第二核酸分子的平端的互补序列杂交，并且拓扑异构酶可以共价连接ds核酸分子。 该方法比先前用于共价连接核酸序列的方法更简单和更有效。
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公开(公告)号：US07528241B2
公开(公告)日：2009-05-05
申请号：US11053187
申请日：2005-02-07
摘要： The present invention provides compositions, methods, and kits for covalently linking nucleic acid molecules. The methods include a strand invasion step, and the compositions and kits are useful for performing such methods. For example, a method of covalently linking double stranded (ds) nucleic acid molecules can include contacting a first ds nucleic acid molecule, which has a topoisomerase linked to a 3′ terminus of one end and has a single stranded 5′ overhang at the same end, with a second ds nucleic acid molecule having a blunt end, such that the 5′ overhang can hybridize to a complementary sequence of the blunt end of the second nucleic acid molecule, and the topoisomerase can covalently link the ds nucleic acid molecules. The methods are simpler and more efficient than previous methods for covalently linking nucleic acid sequences, and the compositions and kits facilitate practicing the methods, including methods of directionally linking two or more ds nucleic acid molecules.
摘要翻译： 本发明提供用于共价连接核酸分子的组合物，方法和试剂盒。 所述方法包括线入侵步骤，组合物和试剂盒对于进行这些方法是有用的。 例如，共价连接双链（ds）核酸分子的方法可以包括使第一个ds核酸分子接触，该第一个ds核酸分子具有与一个末端的3'末端连接的拓扑异构酶并且具有相同的单链（5'）突出端 末端，具有平末端的第二ds核酸分子，使得5'突出端可以与第二核酸分子的平端的互补序列杂交，并且拓扑异构酶可以共价连接ds核酸分子。 该方法比先前用于共价连接核酸序列的方法更简单和更有效，并且组合物和试剂盒有助于实施该方法，包括定向连接两个或多个ds核酸分子的方法。
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5.发明申请公开(公告)号：US20090326208A1
公开(公告)日：2009-12-31
申请号：US12112649
申请日：2008-04-30
申请人： John Carrino , James Fan , Robert P. Bennett , Jonathan D. Chesnut , Martin A. Gleeson , Knut R. Madden
发明人： John Carrino , James Fan , Robert P. Bennett , Jonathan D. Chesnut , Martin A. Gleeson , Knut R. Madden
CPC分类号： C12N15/10 , C12N9/90 , C12N15/111 , C12N15/64 , C12N15/66 , C12N2310/14 , C12N2330/30 , C12P19/34 , C12Q1/6855 , C12Y599/01002 , C12Q2521/519
摘要： A method of generating a double stranded (ds) recombinant nucleic acid molecule covalently linked in both strands by contacting two or more ds nucleotide sequences with a topoisomerase under conditions such that both termini of at least one end of a first ds nucleotide sequence are covalently linked by the topoisomerase to both termini of at least one end of a second ds nucleotide sequence is provided. Also provided is a method for generating a ds recombinant nucleic acid molecule covalently linked in one strand, by contacting two or more ds nucleotide sequences with a type IA topoisomerase under conditions such that one strand, but not both strands, of one or both ends of a first ds nucleotide sequence are covalently linked by the topoisomerase. Compositions for performing such methods, and compositions generated from such methods also are provided, as are kits containing components useful for conveniently practicing the methods.
摘要翻译： 通过使两个或多个ds核苷酸序列与拓扑异构酶接触的条件使得在第一个ds核苷酸序列的至少一个末端的两个末端共价连接的情况下，产生双链（ds）重组核酸分子共价连接的双链（ds）重组核酸分子的方法 提供了拓扑异构酶至第二个ds核苷酸序列的至少一个末端的两个末端。 还提供了一种用于产生共价连接在一条链中的ds重组核酸分子的方法，其通过使两个或更多个ds核苷酸序列与IA型拓扑异构酶接触，使得一个或两个末端的一条链但不是两条链 第一个ds核苷酸序列由拓扑异构酶共价连接。 还提供了用于进行这些方法的组合物，以及由这些方法产生的组合物，以及包含用于方便地实施该方法的组分的试剂盒。
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6.发明授权Compositions and methods for rapidly generating recombinant nucleic acid molecules 有权用于快速产生重组核酸分子的组合物和方法公开(公告)号：US07033801B2
公开(公告)日：2006-04-25
申请号：US10014128
申请日：2001-12-07
申请人： John Carrino , James Fan , Robert P. Bennett , Jonathan D. Chesnut , Martin A. Gleeson , Knut R. Madden
发明人： John Carrino , James Fan , Robert P. Bennett , Jonathan D. Chesnut , Martin A. Gleeson , Knut R. Madden
IPC分类号： C12P19/34
CPC分类号： C12P19/34 , C12N9/90 , C12N15/10 , C12N15/64 , C12N15/66 , C12Q1/6855 , C12Q2521/519
摘要： A method of generating a double stranded (ds) recombinant nucleic acid molecule covalently linked in both strands by contacting two or more ds nucleotide sequences with a topoisomerase under conditions such that both termini of at least one end of a first ds nucleotide sequence are covalently linked by the topoisomerase to both termini of at least one end of a second ds nucleotide sequence is provided. Also provided is a method for generating a ds recombinant nucleic acid molecule covalently linked in one strand, by contacting two or more ds nucleotide sequences with a type IA topoisomerase under conditions such that one strand, but not both strands, of one or both ends of a first ds nucleotide sequence are covalently linked by the topoisomerase. Compositions for performing such methods, and compositions generated from such methods also are provided, as are kits containing components useful for conveniently practicing the methods.
摘要翻译： 通过使两个或多个ds核苷酸序列与拓扑异构酶接触的条件使得在第一个ds核苷酸序列的至少一个末端的两个末端共价连接的情况下，产生在两条链中共价连接的双链（ds）重组核酸分子的方法 提供了拓扑异构酶至第二个ds核苷酸序列的至少一个末端的两个末端。 还提供了一种用于产生共价连接在一条链中的ds重组核酸分子的方法，其通过使两个或更多个ds核苷酸序列与IA型拓扑异构酶接触，使得一个或两个末端的一条链但不是两条链 第一个ds核苷酸序列由拓扑异构酶共价连接。 还提供了用于进行这些方法的组合物，以及由这些方法产生的组合物，以及包含用于方便地实施该方法的组分的试剂盒。
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7.发明申请公开(公告)号：US20110046201A1
公开(公告)日：2011-02-24
申请号：US12112803
申请日：2008-04-30
IPC分类号： A61K31/713 , C12P19/34 , C12N5/10 , C12N15/63
CPC分类号： C12P19/34 , C07H21/04 , C12N9/90 , C12N15/10 , C12N15/111 , C12N15/64 , C12N15/66 , C12N15/86 , C12N2310/111 , C12N2310/14 , C12N2330/30 , C12N2740/16043 , C12N2800/30 , C12N2800/70 , C12Y599/01
摘要： The present invention is in the fields of biotechnology and molecular biology. More particularly, the present invention relates to cloning or subcloning one or more nucleic acid molecules comprising one or more type IIs restriction enzyme recognition sites. The present invention also embodies cloning such nucleic acid molecules using recombinational cloning methods such as those employing recombination sites and recombination proteins. The present invention also relates to nucleic acid molecules (including RNA and iRNA), as well as proteins, expressed from host cells produced using the methods of the present invention.
摘要翻译： 本发明涉及生物技术和分子生物学领域。 更具体地说，本发明涉及克隆或亚克隆一个或多个包含一个或多个II型限制酶识别位点的核酸分子。 本发明还使用重组克隆方法（例如使用重组位点和重组蛋白的那些）来体现克隆这种核酸分子。 本发明还涉及由使用本发明的方法生产的宿主细胞表达的核酸分子（包括RNA和iRNA）以及蛋白质。
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公开(公告)号：US20120149069A1
公开(公告)日：2012-06-14
申请号：US13401680
申请日：2012-02-21
IPC分类号： C12N15/66
CPC分类号： C12P19/34 , C07H21/04 , C12N9/90 , C12N15/10 , C12N15/111 , C12N15/64 , C12N15/66 , C12N15/86 , C12N2310/111 , C12N2310/14 , C12N2330/30 , C12N2740/16043 , C12N2800/30 , C12N2800/70 , C12Y599/01
摘要： The present invention is in the fields of biotechnology and molecular biology. More particularly, the present invention relates to cloning or subcloning one or more nucleic acid molecules comprising one or more type IIs restriction enzyme recognition sites. The present invention also embodies cloning such nucleic acid molecules using recombinational cloning methods such as those employing recombination sites and recombination proteins. The present invention also relates to nucleic acid molecules (including RNA and iRNA), as well as proteins, expressed from host cells produced using the methods of the present invention.
摘要翻译： 本发明涉及生物技术和分子生物学领域。 更具体地说，本发明涉及克隆或亚克隆一个或多个包含一个或多个II型限制酶识别位点的核酸分子。 本发明还使用重组克隆方法（例如使用重组位点和重组蛋白的那些）来体现克隆这种核酸分子。 本发明还涉及由使用本发明的方法生产的宿主细胞表达的核酸分子（包括RNA和iRNA）以及蛋白质。
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公开(公告)号：US07078501B2
公开(公告)日：2006-07-18
申请号：US09792875
申请日：2001-02-23
申请人： John A. Heyman , Gabor Szalai , Michael Felder , Knut R. Madden
发明人： John A. Heyman , Gabor Szalai , Michael Felder , Knut R. Madden
CPC分类号： C12Q1/6869 , C12N15/10 , C12Q1/6855 , C12Q2521/519 , C12Q2525/191 , C12Q2531/113
摘要： A Vaccinia topoisomerase-adapted linker, which contains an oligonucleotide primer binding site of known sequence, useful for specifically joining the linker to the end of a polynucleotide of unknown sequence and allowing subsequent PCR amplification of the polynucleotide or DNA is provided. Kits containing the invention Vaccinia topoisomerase-adapted linker and one or more linker-specific oligonucleotides for annealing to the linker in PCR are also provided. In addition, the invention provides methods for using the invention linkers in linker-mediated PCR amplification procedures for isolation and optional sequencing of isolated PCR amplification products.
摘要翻译： 提供了一种具有已知序列的寡核苷酸引物结合位点的牛痘拓扑异构酶适配接头，其可用于将接头特异性连接到未知序列的多核苷酸的末端并允许随后的多核苷酸或DNA的PCR扩增。 还提供了含有本发明的Vaccinia拓扑异构酶修饰接头和用于在PCR中对接头退火的一个或多个接头特异性寡核苷酸的试剂盒。 此外，本发明提供了使用本发明接头在接头介导的PCR扩增程序中用于分离和任选测序分离的PCR扩增产物的方法。
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10.发明申请COMPOSITIONS AND METHODS FOR PREPARING SHORT RNA MOLECULES AND OTHER NUCLEIC ACIDS 审中-公开用于制备短链RNA分子和其他核酸的组合物和方法公开(公告)号：US20120108804A1
公开(公告)日：2012-05-03
申请号：US13190325
申请日：2011-07-25
申请人： Knut R. Madden , Adam N. Harris , Karl H. Hecker , Byung-in Lee
发明人： Knut R. Madden , Adam N. Harris , Karl H. Hecker , Byung-in Lee
IPC分类号： C07H21/02
CPC分类号： C07H21/02 , B01D15/3804 , C12N15/111 , C12N2330/31
摘要： The invention provides methods of preparing nucleic acids, such as RNA molecules, of a defined size or range of sizes. The invention provides compositions, methods and kits for use in the production and preparation of small RNA molecules (including without limitation micro-RNA, siRNA, d-siRNA and e-siRNA) and other nucleic acids of various sizes.
摘要翻译： 本发明提供了制备具有规定尺寸或尺寸范围的核酸如RNA分子的方法。 本发明提供了用于生产和制备小RNA分子（包括但不限于微RNA，siRNA，d-siRNA和e-siRNA）和各种尺寸的其它核酸的组合物，方法和试剂盒。
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