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1. 发明申请

US20100184187A1 IN VITRO RECOMBINATION METHOD 有权
标题翻译：体外重建方法
公开(公告)号：US20100184187A1
公开(公告)日：2010-07-22
申请号：US12750571
申请日：2010-03-30
申请人： Lei YOUNG , Hamilton O. Smith , Daniel Glenn Gibson
发明人： Lei YOUNG , Hamilton O. Smith , Daniel Glenn Gibson
IPC分类号： C12N9/12
CPC分类号： C12N15/902 , C12N9/1252 , C12N9/22 , C12N9/93 , C12N15/10 , C12N15/64 , C12N15/66 , C12P19/34 , C12Q1/62 , C12Q1/686 , C12Q1/6862 , C12Q2521/501 , C12Q2522/101 , C12Y207/07007 , C12Y301/11003 , C12Y605/01001
摘要： The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonuclease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.
摘要翻译：本发明涉及例如使用分离的蛋白质试剂连接目标的两个双链（ds）DNA分子的体外方法，其中第一DNA分子的远端区域和第二DNA分子的近端区域共享一个包括使反应混合物中的两个DNA分子与（a）非进程性5'核酸外切酶接触; （b）加速核酸退火的单链DNA结合蛋白（SSB）; （c）非链置换DNA聚合酶; 和（d）连接酶，在有效连接两个DNA分子以形成完整双链DNA分子的条件下，其中保留序列同一性区域的单拷贝。该方法允许以预定的顺序和取向连接多个DNA片段，而不使用限制酶。

2. 发明授权

US07723077B2 In vitro recombination method 有权
标题翻译：体外重组法
公开(公告)号：US07723077B2
公开(公告)日：2010-05-25
申请号：US11502746
申请日：2006-08-11
申请人： Lei Young , Hamilton O. Smith , Daniel Glenn Gibson
发明人： Lei Young , Hamilton O. Smith , Daniel Glenn Gibson
IPC分类号： C12P19/34 , C12N9/00 , C12N15/00
CPC分类号： C12N15/902 , C12N9/1252 , C12N9/22 , C12N9/93 , C12N15/10 , C12N15/64 , C12N15/66 , C12P19/34 , C12Q1/62 , C12Q1/686 , C12Q1/6862 , C12Q2521/501 , C12Q2522/101 , C12Y207/07007 , C12Y301/11003 , C12Y605/01001
摘要： The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonculease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.
摘要翻译：本发明涉及例如使用分离的蛋白质试剂连接目标的两个双链（ds）DNA分子的体外方法，其中第一DNA分子的远端区域和第二DNA分子的近端区域共享一个包括使反应混合物中的两个DNA分子与（a）非进程性5'外切酶接触; （b）加速核酸退火的单链DNA结合蛋白（SSB）; （c）非链置换DNA聚合酶; 和（d）连接酶，在有效连接两个DNA分子以形成完整双链DNA分子的条件下，其中保留序列同一性区域的单拷贝。该方法允许以预定的顺序和取向连接多个DNA片段，而不使用限制酶。

3. 发明授权

US09534251B2 In vitro recombination method 有权
标题翻译：体外重组法
公开(公告)号：US09534251B2
公开(公告)日：2017-01-03
申请号：US12750571
申请日：2010-03-30
申请人： Lei Young , Hamilton O. Smith , Daniel Glenn Gibson
发明人： Lei Young , Hamilton O. Smith , Daniel Glenn Gibson
IPC分类号： C12N9/00 , C12Q1/68 , C12N15/66 , C12N15/10 , C12N15/64 , C12P19/34
CPC分类号： C12N15/902 , C12N9/1252 , C12N9/22 , C12N9/93 , C12N15/10 , C12N15/64 , C12N15/66 , C12P19/34 , C12Q1/62 , C12Q1/686 , C12Q1/6862 , C12Q2521/501 , C12Q2522/101 , C12Y207/07007 , C12Y301/11003 , C12Y605/01001
摘要： The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonuclease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.
摘要翻译：本发明涉及例如使用分离的蛋白质试剂连接目标的两个双链（ds）DNA分子的体外方法，其中第一DNA分子的远端区域和第二DNA分子的近端区域共享一个包括使反应混合物中的两个DNA分子与（a）非进程性5'核酸外切酶接触; （b）加速核酸退火的单链DNA结合蛋白（SSB）; （c）非链置换DNA聚合酶; 和（d）连接酶，在有效连接两个DNA分子以形成完整双链DNA分子的条件下，其中保留序列同一性区域的单拷贝。该方法允许以预定的顺序和取向连接多个DNA片段，而不使用限制酶。

4. 发明授权

US08435736B2 Method for in vitro recombination 有权
标题翻译：体外重组方法
公开(公告)号：US08435736B2
公开(公告)日：2013-05-07
申请号：US12820861
申请日：2010-06-22
申请人： Daniel Glenn Gibson , Hamilton O. Smith
发明人： Daniel Glenn Gibson , Hamilton O. Smith
IPC分类号： C12Q1/68 , C12P19/34 , C07H21/02 , C07H21/04
CPC分类号： C12N9/1252 , C12N15/10 , C12N15/102 , C12N15/1031 , C12N15/64 , C12N15/66 , C12P19/34
摘要： The present invention relates to an in vitro method, using isolated protein reagents, for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest.
摘要翻译：本发明涉及一种使用分离的蛋白质试剂连接两个感兴趣的双链（ds）DNA分子的体外方法，其中第一DNA分子的远端区域和第二DNA分子的近端区域共享一个区域的序列同一性。该方法允许以预定的顺序和取向连接多个DNA片段，而不使用限制酶。其可以用于例如合成产生的感兴趣的基因或基因组的亚片段。

5. 发明授权

US07776532B2 Method for in vitro recombination 有权
标题翻译：体外重组方法
公开(公告)号：US07776532B2
公开(公告)日：2010-08-17
申请号：US11502624
申请日：2006-08-11
申请人： Daniel Glenn Gibson , Hamilton O. Smith
发明人： Daniel Glenn Gibson , Hamilton O. Smith
IPC分类号： C12Q1/68 , C12P19/34 , C07H21/02 , C07H21/04
CPC分类号： C12N9/1252 , C12N15/10 , C12N15/102 , C12N15/1031 , C12N15/64 , C12N15/66 , C12P19/34
摘要： The present invention relates, e.g., to an in vitro method, using isolated protein reagents, for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising (a) chewing back the DNA molecules with an enzyme having an exonuclease activity, to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence identity to hybridize specifically to each other; (b) specifically annealing the single-stranded overhangs; and (c) repairing single-stranded gaps in the annealed DNA molecules and sealing the nicks thus formed (ligating the nicked DNA molecules). The region of sequence identity generally comprises at least 20 non-palindromic nucleotides (nt), e.g., at least about 40 non-palindromic nt. In some embodiments of the invention, about 5% PEG is present during all steps of the reaction, and/or the repair reaction is achieved with Taq DNA polymerase and a compatible ligase, such as Taq DNA ligase. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest.
摘要翻译：本发明涉及例如使用分离的蛋白质试剂连接两个目的双链（ds）DNA分子的体外方法，其中第一DNA分子的远端区域和第二DNA分子的近端区域共享序列同一性区域，其包括（a）用具有核酸外切酶活性的酶咀嚼DNA分子，以产生每个DNA分子的单链突出部分，其含有足够长度的序列同一性区域以与彼此; （b）具体退火单链突出端; 和（c）修复退火的DNA分子中的单链间隙并密封由此形成的切口（连接有缺口的DNA分子）。序列同一性区域通常包含至少20个非回文序列核苷酸（nt），例如至少约40个非回文性核苷酸。在本发明的一些实施方案中，在反应的所有步骤期间存在约5％的PEG，和/或用Taq DNA聚合酶和相容连接酶如Taq DNA连接酶实现修复反应。该方法允许以预定的顺序和取向连接多个DNA片段，而不使用限制酶。其可以用于例如合成产生的感兴趣的基因或基因组的亚片段。

6. 发明授权

US09593329B2 Assembly of large nucleic acids 有权
标题翻译：装配大型核酸
公开(公告)号：US09593329B2
公开(公告)日：2017-03-14
申请号：US12247126
申请日：2008-10-07
申请人： Daniel G. Gibson , Lei Young , John I. Glass , Gwynedd A. Benders , J. Craig Venter , Clyde A. Hutchison, III , Hamilton O. Smith
发明人： Daniel G. Gibson , Lei Young , John I. Glass , Gwynedd A. Benders , J. Craig Venter , Clyde A. Hutchison, III , Hamilton O. Smith
IPC分类号： C12N15/10 , C12N15/81
CPC分类号： C12N15/1093 , C12N15/81
摘要： A method to assemble any desired nucleic acid molecule by combining cassettes in vitro to form assemblies which are further combined in vivo, or by assembling large numbers of DNA fragments by recombination in a yeast culture to obtain desired DNA molecules of substantial size is described.
摘要翻译：描述了通过在体外组合盒来组装任何所需核酸分子以形成在体内进一步组合的组装或通过在酵母培养物中通过重组组装大量DNA片段以获得所需的相当大小的DNA分子来组装任何所需核酸分子的方法。

7. 发明授权

US09434974B2 Installation of genomes or partial genomes into cells or cell-like systems 有权
标题翻译：将基因组或部分基因组安装到细胞或细胞样系统中
公开(公告)号：US09434974B2
公开(公告)日：2016-09-06
申请号：US11644713
申请日：2006-12-22
申请人： John I. Glass , Lei Young , Carole Lartigue , Nacyra Assad-Garcia , Hamilton O. Smith , Clyde A. Hutchison , J. Craig Venter
发明人： John I. Glass , Lei Young , Carole Lartigue , Nacyra Assad-Garcia , Hamilton O. Smith , Clyde A. Hutchison , J. Craig Venter
IPC分类号： C12N15/74 , C12N15/63 , C12N15/10 , C12R1/35 , C12P21/02
CPC分类号： C12P21/02 , C12N2510/00
摘要： A method is provided for introducing a genome into a cell or cell-like system. The introduced genome may occur in nature, be manmade with or without automation, or may be a hybrid of naturally occurring and manmade materials. The genome is obtained outside of a cell with minimal damage. Materials such as a proteins, RNAs, polycations, nucleoid condensation proteins, or gene translation systems may accompany the genome. The genome is installed into a naturally occurring cell or into a manmade cell-like system. A cell-like system or synthetic cell resulting from the practice of the provided method may be designed and used to yield gene-expression products, such as desired proteins. By enabling the synthesis of cells or cell-like systems comprising a wide variety of genomes, accompanying materials and membrane types, the provided method makes possible a broader field of experimentation and bioengineering than has been available using prior art methods.
摘要翻译：提供了将基因组导入细胞或细胞样系统的方法。引入的基因组可以在自然界中发生，在有或没有自动化的情况下是人造的，或者可以是天然存在的和人造的材料的混合物。基因组以最小的损伤在细胞外获得。材料如蛋白质，RNAs，聚阳离子，核仁缩合蛋白或基因翻译系统可能伴随着基因组。将基因组安装到天然存在的细胞或人造细胞样系统中。可以设计并使用由所提供的方法的实践产生的细胞样系统或合成细胞来产生基因表达产物，例如所需的蛋白质。通过实现包含各种各样的基因组，伴随材料和膜类型的细胞或细胞样系统的合成，所提供的方法可能比使用现有技术方法可用的更广泛的实验和生物工程领域。

8. 发明授权

US08968999B2 Methods for in vitro joining and combinatorial assembly of nucleic acid molecules 有权
标题翻译：核酸分子的体外连接和组合装配方法
公开(公告)号：US08968999B2
公开(公告)日：2015-03-03
申请号：US12371543
申请日：2009-02-13
申请人： Daniel G. Gibson , Hamilton O. Smith , Clyde A. Hutchison , Lei Young , J. Craig Venter
发明人： Daniel G. Gibson , Hamilton O. Smith , Clyde A. Hutchison , Lei Young , J. Craig Venter
IPC分类号： C12Q1/68 , C12P19/34 , C07H21/02 , C12N15/66 , C12N15/10 , C12N15/64
CPC分类号： C12P19/34 , C12N15/10 , C12N15/1027 , C12N15/64 , C12N15/66
摘要： The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.
摘要翻译：本发明涉及在体外连接两个或多个目的双链（ds）或单链（ss）DNA分子的方法，其中第一DNA分子的远端区域和第二DNA分子的近端区域每对共享序列同一性区域。该方法允许以预定顺序和取向连接大量DNA片段，而不使用限制酶。其可以用于例如合成产生的感兴趣的基因或基因组的亚片段。还公开了用于执行该方法的套件。连接DNA分子的方法可用于产生组合文库，其可用于产生例如通过密码子优化，基因优化和途径优化的最佳蛋白质表达。

9. 发明申请

US20120053087A1 METHODS FOR IN VITRO JOINING AND COMBINATORIAL ASSEMBLY OF NUCLEIC ACID MOLECULES 审中-公开
标题翻译：核酸分子的体外联合和组合组装方法
公开(公告)号：US20120053087A1
公开(公告)日：2012-03-01
申请号：US13035699
申请日：2011-02-25
申请人： Daniel G. Gibson , Hamilton O. Smith , Clyde A. Hutchison , Lei Young , J. Craig Venter
发明人： Daniel G. Gibson , Hamilton O. Smith , Clyde A. Hutchison , Lei Young , J. Craig Venter
IPC分类号： C12P19/34 , C40B40/06 , C07H21/04 , C12N9/12
CPC分类号： C12P19/34 , C12N15/10 , C12N15/1027 , C12N15/64 , C12N15/66
摘要： The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.
摘要翻译：本发明涉及在体外连接两个或多个目的双链（ds）或单链（ss）DNA分子的方法，其中第一DNA分子的远端区域和第二DNA分子的近端区域每对共享序列同一性区域。该方法允许以预定顺序和取向连接大量DNA片段，而不使用限制酶。其可以用于例如合成产生的感兴趣的基因或基因组的亚片段。还公开了用于执行该方法的套件。连接DNA分子的方法可用于产生组合文库，其可用于产生例如通过密码子优化，基因优化和途径优化的最佳蛋白质表达。

10. 发明申请

US20090275086A1 ASSEMBLY OF LARGE NUCLEIC ACIDS 有权
标题翻译：大型核酸成员大会
公开(公告)号：US20090275086A1
公开(公告)日：2009-11-05
申请号：US12247126
申请日：2008-10-07
申请人： Daniel G. Gibson , Lei Young , John I. Glass , Gwynedd A. Benders , J. Craig Venter , Clyde A. Hutchison, III , Hamilton O. Smith
发明人： Daniel G. Gibson , Lei Young , John I. Glass , Gwynedd A. Benders , J. Craig Venter , Clyde A. Hutchison, III , Hamilton O. Smith
IPC分类号： C12P19/34 , C12N1/19 , C07H21/04
CPC分类号： C12N15/1093 , C12N15/81
摘要： A method to assemble any desired nucleic acid molecule by combining cassettes in vitro to form assemblies which are further combined in vivo, or by assembling large numbers of DNA fragments by recombination in a yeast culture to obtain desired DNA molecules of substantial size is described.
摘要翻译：描述了通过在体外组合盒来组装任何所需核酸分子以形成在体内进一步组合的组装或通过在酵母培养物中通过重组组装大量DNA片段以获得所需的相当大小的DNA分子来组装任何所需核酸分子的方法。

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