Techniques for producing site-directed mutagenesis of cloned DNA
    46.
    发明授权
    Techniques for producing site-directed mutagenesis of cloned DNA 失效
    用于产生克隆DNA定点诱变的技术

    公开(公告)号:US5284760A

    公开(公告)日:1994-02-08

    申请号:US764085

    申请日:1991-09-23

    摘要: A method is described whereby new cDNA or RNA sequences can be introduced into or substituted for cDNA in any chosen position without specific sequence requirements using the polymerase chain reaction (PCR). The method entails the use of primers which are complementary to the 3' and 5' ends of the desired sequence to be inserted as well as to the 3' and 5' ends of the chosen site of insertion in the acceptor molecule. The desired sequence is amplified by PCR such that single stranded fragments are produced. The single stranded fragment of the desired sequence is then annealed to a single stranded acceptor molecule at the site of insertion and extended to produce a double stranded molecule. The double stranded molecule is then separated into two strands which are identical except that one of the strands contains the desired sequence inserted at the chosen site. A second double stranded molecule is then generated.

    摘要翻译: 描述了一种方法,其中可以使用聚合酶链式反应(PCR)将新的cDNA或RNA序列引入任何选定位置或用任何特定序列要求代替cDNA。 该方法需要使用与待插入的所需序列的3'和5'末端互补的引物以及在受体分子中所选插入位点的3'和5'末端。 通过PCR扩增所需的序列,从而产生单链片段。 然后将所需序列的单链片段在插入位点退火至单链受体分子并延伸以产生双链分子。 然后将双链分子分成两条相同的链,除了一条链含有插入所选位点的所需序列外。 然后产生第二双链分子。