title: An improved PCR-based method for site directed mutagenesis using megaprimers.
abstract: An improved protocol for site-directed mutagenesis based on the two-step polymerase chain reaction (PCR) megaprimer method is described. Compared to previously published protocols, the protocol described in this article ensures consistently a success rate of at least 85% with essentially no introduction of unwanted secondary mutations. The essential features of this protocol include an optimization of the template-primer amounts and ratio that allows the use of a reduced number of PCR cycles and the use of proof-reading thermostable DNA polymerases.
authors.affiliation: Department of Molecular Biology, Odense University, Odense, Denmark.
authors.forename: J
authors.initials: J
authors.lastname: Brøns-Poulsen
authors.forename: N E
authors.initials: NE
authors.lastname: Petersen
authors.forename: M
authors.initials: M
authors.lastname: Hørder
authors.forename: K
authors.initials: K
authors.lastname: Kristiansen
mesh_terms: D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D011163:Hydroxymethylbilane Synthase; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase