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    SYNTHESIS OF ERROR-MINIMIZED NUCLEIC ACID MOLECULES
    41.
    发明授权
    SYNTHESIS OF ERROR-MINIMIZED NUCLEIC ACID MOLECULES 有权
    以最小的错误数量的核酸分子的合成

    公开(公告)号：EP1963525B1

    公开(公告)日：2013-11-20

    申请号：EP06844856.2

    申请日：2006-12-04

    申请人： Synthetic Genomics, Inc.

    发明人： YOUNG, Lei

    IPC分类号： C12N15/10

    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.

    Method for in vitro recombination
    47.
    发明公开
    Method for in vitro recombination 有权
    Verfahren zur体外重组

    公开(公告)号：EP2239327A2

    公开(公告)日：2010-10-13

    申请号：EP10168787.9

    申请日：2006-08-11

    申请人： Synthetic Genomics, Inc.

    发明人： Gibson, Daniel Glenn ,  Smith, Hamilton, O.

    IPC分类号： C12N15/09 ,  C12N15/00

    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 for joining a first set of double-stranded (ds) DNA molecules, comprising:
    (a) providing two or more dsDNA molecules to be joined, wherein, for each pair of dsDNA molecules to be joined, the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence homology comprising at least about 20 non-palindromic nucleotides;
    (b) treating the provided dsDNA molecules with a substantially purified enzyme having 5'-3' exonuclease activity, whereby single-stranded overhanging portions are generated in the dsDNA molecules by 5'-3' exonuclease digestion, wherein each overhanging portion contains the region of homology or a portion thereof sufficient to specifically hybridize to the overhanging portion in the other molecule of the pair;
    (c) incubating the DNA molecules generated in step (b), under conditions whereby they anneal through the overhanging portions; and
    (d) treating the annealed molecules with a substantially purified DNA polymerase and a substantially purified compatible ligase, under conditions whereby remaining single-stranded gap(s) are filled in by the polymerase and nicks are sealed by the ligase;

    thereby joining the dsDNA molecules, wherein the method is carried out in the presence of a crowding agent ,e.g. about 5% PEG.

    摘要翻译： 本发明涉及一种用于连接第一组双链（ds）DNA分子的体外方法，其包括：（a）提供两个或更多个待连接的dsDNA分子，其中，对于待连接的每对双链DNA分子 所述第一DNA分子的远侧区域和所述第二DNA分子的近端区域共享包含至少约20个非回归性核苷酸的序列同源性区域; （b）用具有5'-3'核酸外切酶活性的基本上纯化的酶处理所提供的dsDNA分子，由此通过5'-3'外切核酸酶消化在dsDNA分子中产生单链突出部分，其中每个悬伸部分包含区域 的同源性或其一部分足以与该对的另一个分子中的突出部分特异性杂交; （c）在步骤（b）中产生的DNA分子在通过悬伸部分退火的条件下孵育; 和（d）在基本上纯化的DNA聚合酶和基本上纯化的相容性连接酶的条件下处理退火的分子，其中由聚合酶填充剩余的单链间隙，并且切口用连接酶密封; 从而连接dsDNA分子，其中所述方法在拥挤剂存在下进行，例如， 约5％PEG。