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1. 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。

2. 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。

3. 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。

4. 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。

5. 发明申请

US20070269862A1 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：US20070269862A1
公开(公告)日：2007-11-22
申请号：US11644713
申请日：2006-12-22
申请人： John Glass , Lei Young , Carole Lartigue , Nacyra Assad-Garcia , Hamilton Smith , Clyde Hutchison , J. Venter
发明人： John Glass , Lei Young , Carole Lartigue , Nacyra Assad-Garcia , Hamilton Smith , Clyde Hutchison , J. Venter
IPC分类号： C12N15/87 , C12N5/06 , C12P21/04
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。

6. 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。

7. 发明授权

US07704690B2 Synthesis of error-minimized nucleic acid molecules 有权
标题翻译：错误最小化核酸分子的合成
公开(公告)号：US07704690B2
公开(公告)日：2010-04-27
申请号：US11633686
申请日：2006-12-04
申请人： Lei Young
发明人： Lei Young
IPC分类号： C12Q1/68 , C12P19/34
CPC分类号： C12N15/1093 , C12N15/1027
摘要： A method is provided for synthesis of error-minimized nucleic acid molecules. Oligonucleotides intended to have fragments of a desired, full-length nucleotide sequence, and optionally containing other desired nucleotides, such as nucleotides for binding the oligonucleotides to a substrate, are obtained. Oligonucleotides for both strands of the desired, full-length sequence may be obtained. The oligonucleotides are amplified and assembled into a first set of molecules intended to have the desired, full-length nucleotide sequence. The first set of molecules is denatured and annealed to form a second set of molecules intended to have the desired, full-length nucleotide sequence. The second set of molecules is cut into smaller segments, for example, by mixing the molecules with endonucleases that form blunt cuts in the second set of molecules where there are sequence errors, as well as randomly along the molecules. The smaller segments are assembled into a set of molecules intended to have the desired, full-length nucleotide sequence. By promoting cutting of the molecules in this manner near the end of the nucleic acid molecule synthesis process, a set of full-length molecules can be obtained with fewer nucleotide sequence errors than can be achieved with other methods.
摘要翻译：提供了用于合成错误最小化的核酸分子的方法。旨在具有期望的全长核苷酸序列的片段和任选地含有其它所需核苷酸的寡核苷酸，例如将寡核苷酸与底物结合的核苷酸。可以获得所需全长序列的两条链的寡核苷酸。将寡核苷酸扩增并组装成旨在具有所需的全长核苷酸序列的第一组分子。第一组分子被变性和退火以形成旨在具有所需的全长核苷酸序列的第二组分子。第二组分子被切割成更小的片段，例如通过将分子与在其中存在序列错误的第二组分子中形成钝角切割的内切核酸酶以及沿分子随机混合。较小的片段被组装成一组旨在具有所需的全长核苷酸序列的分子。通过在核酸分子合成过程结束时以这种方式促进分子的切割，可以获得比用其它方法可以获得的更少的核苷酸序列错误的全长分子。

8. 发明申请

US20070037197A1 In vitro recombination method 有权
标题翻译：体外重组法
公开(公告)号：US20070037197A1
公开(公告)日：2007-02-15
申请号：US11502746
申请日：2006-08-11
申请人： Lei Young , Hamilton Smith , Daniel Gibson
发明人： Lei Young , Hamilton Smith , Daniel Gibson
IPC分类号： C12Q1/68 , 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′ 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。

9. 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。

10. 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。

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