title: REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering.
abstract: Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient.
authors.affiliation: Department of Physiology, Institute of Biomedicine, University of Turku, Turku, Finland.
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Trehan
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of Lublin, Lublin, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Kiełbus
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of Lublin, Lublin, Poland.
authors.forename: Jakub
authors.initials: J
authors.lastname: Czapinski
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of Lublin, Lublin, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Stepulak
authors.affiliation: Department of Physiology, Institute of Biomedicine, University of Turku, Turku, Finland.|Department of Surgery and Cancer, Institute of Reproductive and Developmental Biology, Hammersmith Campus, Imperial College London, London, United Kingdom.
authors.forename: Ilpo
authors.initials: I
authors.lastname: Huhtaniemi
authors.affiliation: Department of Physiology, Institute of Biomedicine, University of Turku, Turku, Finland.|Faculty of Natural Sciences and Technology, Åbo Akademi University, Turku, Finland.|Department of Biochemistry and Molecular Biology, Medical University of Lublin, Lublin, Poland.
authors.forename: Adolfo
authors.initials: A
authors.lastname: Rivero-Müller
mesh_terms: D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D005818:Genetic Engineering; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic