title: Site-directed mutagenesis by double polymerase chain reaction.
abstract: A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile 36688-0002, USA.
authors.forename: S
authors.initials: S
authors.lastname: Barik
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers