title: An In Vitro Single-Primer Site-Directed Mutagenesis Method for Use in Biotechnology.
abstract: Site-directed mutagenesis is a powerful method to introduce mutation(s) into DNA sequences. A number of methods have been developed over the years with a main goal being to create a high number of mutant genes. The single-mutagenic primer method for site-directed mutagenesis is the most direct method that yields mutant genes in about 25-50 % of transformants in a robust, low-cost reaction. The supercompetent XL10-Gold bacteria used in the Stratagene protocol carry a phage, which may be a problem for some applications; however, in our single-mutagenic primer method the supercompetent bacteria are not needed. A thermostable DNA polymerase with high fidelity and processivity, such as Phusion DNA polymerase, is required for our optimized procedure to avoid extra mutation(s) and enhance mutagenic efficiency.
authors.affiliation: Marine Science & Technology Institute, Department of Environmental Science and Engineering, Yangzhou University, No. 196 Huayang West Road, Yangzhou City, Hanjiang District, Jiangsu Province, 225127, China.
authors.forename: Yanchao
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Marine Science & Technology Institute, Department of Environmental Science and Engineering, Yangzhou University, No. 196 Huayang West Road, Yangzhou City, Hanjiang District, Jiangsu Province, 225127, China. lkzhang@yzu.edu.cn.
authors.forename: Likui
authors.initials: L
authors.lastname: Zhang
mesh_terms: D001419:Bacteria / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D017931:DNA Primers / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase
keywords: Competent Escherichia coli; DpnI digestion; Single-mutagenic primer method; Site-directed mutagenesis; Thermostable DNA polymerase