EFFICIENT SITE-DIRECTED INVITRO MUTAGENESIS USING PHAGEMID VECTORS

  • 1 March 1989
    • journal article
    • research article
    • Vol. 7  (3) , 282-+
Abstract
Several methods have been developed that enhance the efficiency of in vitro, site-directed mutagenesis. Kunkel (8.9) has developed a method which uses a strong selection for the mutated strand and, hence, is highly efficient, but yet simple and rapid. This method orginally used M13 phage as the vector. In this paper, we describe a refinement of this method using phagemid vectors, which combine the advantages of plasmids (such as high copy number and stability of cloned DNA) with the single-stranded DNA generating capability of M13 phage. We demonstrate that high efficiency of mutant production can be obtained with these vectors. We also analyzed by sequencing 11 mutated clones and found no second-site mutations, suggesting that alterations other than the site-directed rarely occur in our system.