title: A "mesmer"izing new approach to site-directed mutagenesis in large transformation-ready constructs: Mutagenesis via Serial Small Mismatch Recombineering.
abstract: Creating designer mutations in large genes is a challenge. Size limitations imposed by site-directed mutagenesis (SDM), coupled with the paucity of unique restriction enzyme sites, make subsequent cloning of these constructs extremely difficult. "Mutagenesis via Serial Small Mismatch Recombineering" (MSSMR) combines sequential recombineering steps with SDM to create seamless, pre-specified mutations as small as a single base pair. We demonstrate the simultaneous cloning of wild type and mutant constructs of a > 30 kb gene directly into attB transformation vectors. No post-transformation manipulations are required, and because the technique relies on recombineering methods, addition of undesired mutations via PCR is minimized.
authors.affiliation: Department of Biology, University of Iowa, Iowa City, IA, USA.
authors.forename: Julie S
authors.initials: JS
authors.lastname: Jacobs
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Hong
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Eberl
mesh_terms: D000818:Animals; D020137:Base Pair Mismatch; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D016297:Mutagenesis, Site-Directed / Q000379:methods*
chemical_list: D029742:Caenorhabditis elegans Proteins; D029721:Drosophila Proteins