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    Method of producing short hairpin library
    46.
    发明申请
    Method of producing short hairpin library 审中-公开
    短发夹文库的生产方法

    公开(公告)号：US20070141594A1

    公开(公告)日：2007-06-21

    申请号：US11546019

    申请日：2006-10-11

    申请人： Biao Luo ,  David Root ,  Xiaoping Yang ,  Gregory Hinkle

    发明人： Biao Luo ,  David Root ,  Xiaoping Yang ,  Gregory Hinkle

    IPC分类号： C40B40/08 ,  C40B50/06

    CPC分类号： C12N15/1093 ,  C12N15/111 ,  C12N2310/14 ,  C12N2310/53 ,  C12N2330/31

    摘要： Described herein is a method of cloning synthetic oligos (including in situ synthesized oligos) into an (one or more) expression vector for library (e.g., shRNA library) production. The oligos are synthesized with one portion of the first stem of the hairpin, followed by a first loop sequence, the complete second stem, a second loop sequence, and finished with the remaining portion of the first stem of the hairpin. The two portions of the first stem anneal to the second stem, juxtaposing the 5′ end close to the 3′ end of the oligo. The methods described herein selected for hairpins with perfectly base-paired stems. After annealing, a ligase is added to the annealed oligos and the base-paired hairpins are preferentially annealed, and ligated, creating closed circular oligos. The now circularized hairpins served as templates for rolling circle amplification using a polymerase with high processivity. One or more primers complementary to the two strands of the amplified double stranded circular hairpins initiate the rolling circle amplification in the presence of a polymerase. Using primers (e.g., a sense and antisense primer), the rolling circle amplification yields double stranded hairpin sequences. These can be digested (e.g., using restriction enzymes) to produce a double-stranded hairpin fragment encoding a single hairpin. The fragment can be cloned into an appropriately digested vector for a variety of uses including expression.

    摘要翻译： 本文描述了将合成寡核苷酸（包括原位合成的寡核苷酸）克隆到（一个或多个）用于文库（例如shRNA文库）生产的表达载体中的方法。 寡核苷酸与发夹的第一茎的一部分合成，随后是第一环序列，完整的第二茎，第二环序列，并用发夹的第一茎的剩余部分完成。 第一茎的两个部分与第二茎退火，将5'末端与寡核苷酸的3'末端并列。 本文所述的方法选择用于具有完全碱基配对茎的发夹。 退火后，将连接酶加入到退火的寡核苷酸中，并且将碱基配对的发夹优先退火并连接，产生闭合的环状寡核苷酸。 现在的圆形发夹作为使用高活性的聚合酶进行滚环扩增的模板。 与扩增的双链环状发夹的两条链互补的一个或多个引物在聚合酶存在下引发滚环扩增。 使用引物（例如有义和反义引物），滚环扩增产生双链发夹序列。 这些可以被消化（例如，使用限制酶）以产生编码单个发夹的双链发夹片段。 可以将片段克隆到适当消化的载体中，用于多种用途，包括表达。