title: A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study.
abstract: PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology.
authors.affiliation: Department of Physiology and Biophysics, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, 5th Floor, Research Tower, Piscataway, NJ 08854-5635, USA.
authors.forename: Jae-Kyun
authors.initials: JK
authors.lastname: Ko
authors.forename: Jianjie
authors.initials: J
authors.lastname: Ma
mesh_terms: D001483:Base Sequence; D002468:Cell Physiological Phenomena; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific / Q000502:physiology*; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific