In vitro insertional mutagenesis with a selectable DNA fragment

摘要: A new method for in vitro insertional mutagenesis of genes cloned Escherichia coli is presented. This simple procedure combines the advantages DNA linker with those vivo transposition mutagenesis. It makes use omega fragment, a 2.0-kb segment consisting an antibiotic resistance gene (the Smr/Spcr R100.1 plasmid) flanked by short inverted repeats carrying transcription and translation termination signals synthetic polylinkers. The fragment inserted into linearized plasmid ligation, recombinant molecules are selected their to streptomycin spectinomycin. terminates RNA protein synthesis prematurely, thus allowing definition mapping both units. Because symmetrical structure omega, same effect obtained insertions either orientation. can be subsequently excised from mutated molecules, leaving behind its flanking restriction site(s).