REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

Abstract

<p> 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 <em>recombineering</em>, which uses bacteria expressing viral recombination proteins. Recently, the use of <em>in vitro</em> seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these <em>in vitro </em>isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes <em>in vitro</em> 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 (<em>R</em>ecombineering of <em>E</em>nds of linearised <em>PLA</em>smids after P<em>CR</em>), for creating mutations (deletions, substitutions and additions) in plasmids by <em>in vivo</em> 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.</p>