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    Tailored multi-site combinatorial assembly
    5.
    发明授权
    Tailored multi-site combinatorial assembly 有权
    定制多地点组合装配

    公开(公告)号：US09476078B2

    公开(公告)日：2016-10-25

    申请号：US12671231

    申请日：2008-07-31

    申请人： Xuqiu Tan

    发明人： Xuqiu Tan

    IPC分类号： C12P19/34 ,  C12N15/10

    CPC分类号： C12P19/34 ,  C12N15/102 ,  C12N15/1024

    摘要： The present invention provides a novel method of producing a plurality of modified polynucleotides having different combinations of various mutations at multiple sites by a tailored multi-site combinatorial assembly, comprising adding at least two or at least three primers to a double stranded template polynucleotide in a single reaction mixture, wherein the primers are not overlapping, and wherein each of the primers comprise at least one mutation different from the other primers, wherein at least one primer is a forward primer that can anneal to a minus strand of the template and at least one primer is a reverse primer that can anneal to a plus strand of the template, and subjecting the reaction mixture to a polymerase extension reaction to yield a plurality of extended modified polynucleotides from the at least three primers. The method can be performed without employing a ligation step prior to transforming the extended modified polynucleotides into a cell. The plurality of extended modified polynucleotides can be treated with an enzyme for destroying the template polynucleotide prior to transforming in to the cell.

    摘要翻译： 本发明提供了一种通过定制的多位点组合组合产生多个具有多个位点的不同突变组合的多个修饰的多核苷酸的新方法，包括向双链模板多核苷酸中添加至少两个或至少三个引物 单反应混合物，其中引物不重叠，并且其中每个引物包含与其它引物不同的至少一个突变，其中至少一个引物是可以与模板的负链退火的至少一个引物，并且至少 一个引物是可以与模板的正链退火的反向引物，并且使反应混合物进行聚合酶延伸反应，以从至少三种引物产生多个延伸的修饰的多核苷酸。 在将延伸的修饰的多核苷酸转化成细胞之前，可以不使用连接步骤进行该方法。 多个延伸修饰的多核苷酸可以在用于在转化到细胞之前破坏模板多核苷酸的酶处理。

    TAILORED MULTI-SITE COMBINATORIAL ASSEMBLY
    6.
    发明申请
    TAILORED MULTI-SITE COMBINATORIAL ASSEMBLY 有权
    定制多地点组合装配

    公开(公告)号：US20100216192A1

    公开(公告)日：2010-08-26

    申请号：US12671231

    申请日：2008-07-31

    申请人： Xuqiu Tan

    发明人： Xuqiu Tan

    IPC分类号： C12P19/34

    CPC分类号： C12P19/34 ,  C12N15/102 ,  C12N15/1024

    摘要： The present invention provides a novel method of producing a plurality of modified polynucleotides having different combinations of various mutations at multiple sites by a tailored multi-site combinatorial assembly, comprising adding at least two or at least three primers to a double stranded template polynucleotide in a single reaction mixture, wherein the primers are not overlapping, and wherein each of the primers comprise at least one mutation different from the other primers, wherein at least one primer is a forward primer that can anneal to a minus strand of the template and at least one primer is a reverse primer that can anneal to a plus strand of the template, and subjecting the reaction mixture to a polymerase extension reaction to yield a plurality of extended modified polynucleotides from the at least three primers. The method can be performed without employing a ligation step prior to transforming the extended modified polynucleotides into a cell. The plurality of extended modified polynucleotides can be treated with an enzyme for destroying the template polynucleotide prior to transforming in to the cell.

    摘要翻译： 本发明提供了一种通过定制的多位点组合组合产生多个具有多个位点的不同突变组合的多个修饰的多核苷酸的新方法，包括向双链模板多核苷酸中添加至少两个或至少三个引物 单反应混合物，其中引物不重叠，并且其中每个引物包含与其它引物不同的至少一个突变，其中至少一个引物是可以与模板的负链退火的至少一个引物，并且至少 一个引物是可以与模板的正链退火的反向引物，并且使反应混合物进行聚合酶延伸反应，以从至少三个引物产生多个延伸的修饰的多核苷酸。 在将延伸的修饰的多核苷酸转化成细胞之前，可以不使用连接步骤进行该方法。 多个延伸修饰的多核苷酸可以在用于在转化到细胞之前破坏模板多核苷酸的酶处理。