Biotechnol. Appl. Biochem. (2008) 49, 175–183 (Printed in Great Britain) doi:10.1042/BA20070045 175 A fast and simple method for simultaneous mixed site-specific mutagenesis of a wide coding sequence Carlo Follo and Ciro Isidoro 1 Dipartimento di Scienze Mediche, Universit` a del Piemonte Orientale ‘A. Avogadro’, Via Solaroli 17, 28100-Novara, Italy Background site-specific mutagenesis at one or mul- tiple sites has recently become an invaluable strategy in functional proteomic studies and genetic engineering. In the present paper we describe a novel PCR-based procedure for site-specific mutagenesis that permits, in a single-step, all three types of nucleotide sequence mutation (deletion, insertion and substitution). The entire procedure is carried out in one tube and takes about 3–4 h. The method utilizes two primers, one of which is phosphorylated at the 5 ′ -terminus, that are designed to directly anneal back-to-back to the target sequence inserted in a plasmid. For the deletion type of mutagenesis (which has virtually no limit to its extent), primers anneal at the ends of the sequence to be deleted. For insertion and substitution types of muta- genesis the primers bear the mutagenic sequences in a tail. The entire circular plasmid, here tested for a max- imum length of 7 kbp, is amplified by inverse PCR. The PCR product incorporates the desired mutagenesis and, after ligation, the plasmid is ready for cloning into bacteria. The method has been proved very efficient for deletions of up to 279 nucleotides, for introducing simultaneous deletions, insertions and substitutions, and for performing alanine scanning over a wide coding region. The procedure is suitable for applications in genetic engineering and for the construction of libraries. Introduction Site-specific mutagenesis is a powerful way of modifying target portions of the DNA sequence that can help to identify regions of interaction with other molecules or, in case of sequence coding for peptides, to explore the structure–function relationship of the protein with regard to its targeting, folding, enzyme activity and post-translational modifications or its interactions with other molecules, such as transporters, inhibitors or substrates. Typical modific- ations that can be obtained by site-specific mutagenesis include generation of cysteine-less proteins, alanine-scanning mutagenesis, codon optimization for heterologous expres- sion, and deletion or insertion of peptide regions (for re- views, see [1–3]). A variety of methods have been proposed for site-specific mutagenesis [4–8], several of them being available as commercial kits. Among these methods, the ones that are PCR-based and that do not require subcloning of the sequence of interest into new plasmids are certainly the most appealing, because of their simplicity and rapidity of execution [9–13]. However, PCR methods for site-directed mutagenesis yield a high percentage of non-mutated or incorrectly mutated clones. Other limitations offered by current methods are represented by the length of the sequence that can be modified, the number and the types of modifications that can be introduced at one time and the rate at which mutagenized plasmids are produced. Here we propose 3SM, a fast and simple method for simultaneous mixed site-specific mutagenesis of a wide non-restricted DNA region within a circular plasmid. It is not mandatory to amplify the template DNA in DAM + (deoxyadenosine methylase + ) bacteria, as the PCR product can be purified from the mix without performing digestion with DpnI (Stratagene, La Jolla, CA, U.S.A.), a restriction endonuclease specific for methylated DNA. The two primers, one of which is 5 ′ -phosphorylated, are designed to anneal back-to-back on the opposing DNA strands of the target region. For substitutions and/or insertions, the mutagenic sequences are incorporated in the tails of the primers. Deletion of nucleotides is achieved by employing primers that anneal at the extremities of the region to be deleted. Mutagenesis is introduced in the plasmid through inverse PCR amplification. Various alternative strategies can be adopted to remove the template DNA and obtain the purified PCR product. The latter is then re-circularized by a DNA ligase reaction and cloned into competent bacteria. This method has been successfully employed for the introduction of a variety of mixed mutations in a wide coding region of a 7 kbp circular plasmid. Key words: alanine scanning, cathepsin D, mixed site-specific mutagenesis, nucleotide sequence mutation, wide coding sequence. Abbreviations used: DAM + , deoxyadenosine methylase + ; 3SM, a fast and simple method for simultaneous mixed site-specific mutagenesis; SLIM, site-directed ligase-independent mutagenesis; T m , ‘melting’ temperature. 1 To whom correspondence should be addressed (e-mail isidoro@med.unipmn.it). C  2008 Portland Press Ltd