A fast and simple method for simultaneous mixed site-specific mutagenesis of a wide coding sequence.

Site-specific mutagenesis at one or multiple sites has recently become an invaluable strategy in functional proteomic studies and genetic engineering.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 to 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 deletion type of mutagenesis (of virtually no limits of extent), primers anneal at the ends of the sequence to be deleted. For insertion and substitution types of mutagenesis, the primers bear the mutagenic sequences in a tail. The entire circular plasmid, here tested for a maximum 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 in bacteria.The method has been proved very efficient to delete up to 279 nucleotides, to introduce simultaneously deletions, insertions and substitutions and to perform the alanine-scanning in a wide coding region. The procedure is suitable for applications in gene engineering and for construction of libraries.