title: Exploring the potential of megaprimer PCR in conjunction with orthogonal array design for mutagenesis library construction.
abstract: Although megaprimer PCR mutagenesis has been used routinely in protein directed evolution, users sometimes encounter technical hurdles, particularly inefficiency during amplification when large fragments are used or the template is difficult to be amplified. Instead of methodology development, here we simply overcome the limitation by optimizing megaprimer PCR conditions via orthogonal array design of the four PCR components in three levels of each: template, primer, Mg(2+) , and dNTPs. For this, only nine PCRs need to be performed. The strategy (termed as OptiMega) was not only successfully applied for the construction of one multiple-site saturation mutagenesis library of halohydrin dehalogenase HheC, which failed to be constructed previously using the standard QuikChange™ protocol, but also expanded the construction of two high-quality random mutagenesis libraries of HheA and HheC. Most importantly, OptiMega offers a quick and simple way of constructing random mutagenesis libraries by eliminating the ligation step. Our results demonstrated that the OptiMega strategy could greatly strengthen the potential of megaprimer PCR mutagenesis for library construction.
authors.affiliation: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China. lixiatang@uestc.edu.cn
authors.forename: Lixia
authors.initials: L
authors.lastname: Tang
authors.forename: Kai
authors.initials: K
authors.lastname: Zheng
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.forename: Huayu
authors.initials: H
authors.lastname: Zheng
authors.forename: Hu
authors.initials: H
authors.lastname: Wang
authors.forename: Chunlei
authors.initials: C
authors.lastname: Song
authors.forename: Hong
authors.initials: H
authors.lastname: Zhou
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers