Being the region unique mutation introduction

摘要

PURPOSE: To conduct more easily a transduction of site-specific mutation in the genetic engineering by thermal modification of a double-stranded DNA vector inserted with a target DNA fragment for site-specific mutation to form a single-stranded DNA. ;CONSTITUTION: Variant primers and selective primers are mixed, so as to be excess in the latter, with a double-stranded DNA vector inserted with a target DNA fragment for site-specific mutation, and this vector is thermally modified and converted into a single-stranded DNA, and both the kinds of primers are mutually hybridized and the gaps between both the kinds of primers are filled through polymerase reaction and ligase reaction, thus preparing the objective double-stranded DNA mutational and perfectly complementary except for the mutational point in the selective sequence. With this DNA, a host Escherichia coli for mustS is transformed and grown, and another host Escherichia coli for sup° is also transformed, and the resultant transformants are cultured in the presence of chloramphenicol Cm or kanamycin Km and clones having mutational DNA are selected. In the figure, R and S denote tolerance and sensitivity, respectively.;COPYRIGHT: (C)1995,JPO