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1. 发明申请

US20100035768A1 METHODS FOR IN VITRO JOINING AND COMBINATORIAL ASSEMBLY OF NUCLEIC ACID MOLECULES 有权
标题翻译：核酸分子的体外联合和组合组装方法
公开(公告)号：US20100035768A1
公开(公告)日：2010-02-11
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： C12P19/34 , C40B40/08 , C07H21/04
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

US20100311126A1 METHOD FOR IN VITRO RECOMBINATION 有权
标题翻译：体外重建方法
公开(公告)号：US20100311126A1
公开(公告)日：2010-12-09
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： C12P19/34
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。

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