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    • 9. 发明公开
    • Efficient directional genetic cloning system
    • Leistungsfähiges系统zur gerichteten genetischen Klonierung
    • EP0773294A2
    • 1997-05-14
    • EP96115534.8
    • 1990-07-30
    • THE UNITED STATES OF AMERICA as represented by the Secretary UNITED STATES DEPARTMENT OF COMMERCE
    • Miki, ToruAaronson, Stuart A.Fleming, Timothy
    • C12N15/16C07K14/71C12N15/10C12N15/85
    • C07K14/71C07K14/82C12N15/1096C12N15/64C12N15/66
    • A highly efficient genetic cloning system is disclosed which is particularly useful for cloning cDNA copies of eukaryotic mRNAS and can direct the orientation of inserts in plasmid composite vectors with large cloning capacities. Cleavage of such vector DNA, by the restriction enzyme SfiI, for example, creates two different non-symmetrical 3' extensions at the ends of vector DNA. Using a linker-primer and an adaptor, cDNA is prepared to have two different sticky ends which can be ligated to those of the vector. When the cDNA fragments and the vector DNAs are mixed, both the molecules can assemble without self-circularization due to base-pairing specificity. This system provides (1) high cloning efficiency (10 7 -10 8 clones/g poly (A) + RNA), (2) low background (more than 90% of the clones contain inserts), (3) directional insertion of cDNA fragments into the vectors, (4) presence of a single insert in each clone, (5) accommodation of long inserts (up to 10kb), (6) a mechanism for rescue of the plasmid part from a λ genome, and (7) a straightforward protocol for library preparation.
    • 公开了一种高效的遗传克隆系统,其特别可用于克隆真核mRNA的cDNA拷贝,并且可以以大克隆能力指导插入片段在质粒复合载体中的取向。 例如,通过限制性酶SfiI切割这种载体DNA,在载体DNA的末端产生两个不同的非对称3'延伸。 使用接头引物和衔接子,制备cDNA具有可以与载体连接的两个不同的粘性末端。 当cDNA片段和载体DNA混合时,由于碱基配对特异性,两个分子都可以组装而不进行自身循环。 该系统提供(1)高克隆效率(10 -7 -10 -10克隆/克聚(A)+ RNA),(2)低背景(超过90%的克隆含有插入片段),( 3)将cDNA片段定向插入到载体中,(4)每个克隆中存在单个插入片段,(5)长插入片段(最多10kb)的适应性,(6)从λ的拯救质粒部分的机制 基因组,和(7)图书馆准备的直接协议。