• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Site-Directed Mutagenesis Using Altered /gb-Lactamase Specificity

        互联网

        541
        Site-directed mutagenesis (SDM) is a powerful tool for the study of gene expression/regulation and protein structure and function. Hutchinson et al. (1 ) developed a general method for the introduction of specific changes in DNA sequence, which involves hybridization of a synthetic oligonucleotide (ON) containing the desired mutation to a single-stranded DNA (ssDNA) target template. Following hybridization, the oligonucleotide is extended with a DNA polymerase to create a double-stranded structure. The heteroduplex DNA is then transformed into an Escherichia coli , in which where both wild type and mutant strands are replicated. In the absence of any selection this method is very inefficient, often resulting in only a few percent of mutants obtained. Various strategies of selection have since been developed, which can increase mutagenesis efficiencies well above the theoretical yield of 50%. The methods of Kunkel (2 ), Eckstein (3 ), and Deng (4 ,5 ) employ negative selection against the wild-type DNA strand, in which the parental DNA is selectively degraded, either by growth in an alternate host strain, or by digestion with a nuclease or restriction enzyme. The methods of Lewis and Thompson (6 ) and Bonsack (7 ) utilize antibiotic resistance to positively select for the mutant DNA strand. This chapter describes a method for the positive selection of mutant strand DNA, which relies on the altered activity of the enzyme β-lactamase against extended spectrum cephalosporins (8 ).
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序