title: Highly efficient one-step PCR-based mutagenesis technique for large plasmids using high-fidelity DNA polymerase.
abstract: Gene mutation plays an important role in molecular biological studies. A highly efficient one-step polymerase chain reaction-based mutagenesis technique for site-directed mutagenesis was developed in this study. One complementary pair of primers was designed that contained the desired mutations in the middle of the primers. The amplification products of mutation were amplified using a high-fidelity DNA polymerase and the original plasmid templates were digested by DpnI. This method was successfully used to introduce mutations in two different-sized plasmids (12 and 6 kb) with high efficiency. The results indicate that this technique can be widely used to introduce any plasmid mutations quickly and efficiently.
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
authors.forename: H
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
authors.forename: R
authors.initials: R
authors.lastname: Ye
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Fudan University, Shanghai, China yuyanss@fudan.edu.cn.
authors.forename: Y Y
authors.initials: YY
authors.lastname: Wang
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004259:DNA-Directed DNA Polymerase