title: Efficient Mutagenesis Independent of Ligation (EMILI).
abstract: Site-directed mutagenesis is one of the most widely used techniques in life sciences. Here we describe an improved and simplified method for introducing mutations at desired sites. It consists of an inverse PCR using a plasmid template and two partially complementary primers. The synthesis step is followed by annealing of the PCR product's sticky ends, which are generated by exonuclease digestion. This method is fast, extremely efficient and cost-effective. It can be used to introduce large insertions and deletions, but also for multiple point mutations in a single step. To show the principle and to prove the efficiency of the method, we present a series of basic mutations (insertions, deletions, point mutations) on pUC19 plasmid DNA.
authors.affiliation: Institute of Chemical Technology Prague, Department of Biochemistry and Microbiology, Technicka 5, 166 28 Prague, Czech Republic.
authors.forename: Tibor
authors.initials: T
authors.lastname: Füzik
authors.affiliation: Institute of Chemical Technology Prague, Department of Biochemistry and Microbiology, Technicka 5, 166 28 Prague, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Ulbrich
authors.affiliation: Institute of Chemical Technology Prague, Department of Biochemistry and Microbiology, Technicka 5, 166 28 Prague, Czech Republic. Electronic address: tomas.ruml@vscht.cz.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Ruml
mesh_terms: D008967:Molecular Biology / Q000379:methods*; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D017354:Point Mutation; D017384:Sequence Deletion
keywords: Fast mutagenesis; Inverse PCR; Large deletion; Mutagenesis; Plasmid-based mutagenesis; T4 DNA polymerase