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Exploring the potential of megaprimer PCR in conjunction with orthogonal array design for mutagenesis library construction
Author(s) -
Tang Lixia,
Zheng Kai,
Liu Yu,
Zheng Huayu,
Wang Hu,
Song Chunlei,
Zhou Hong
Publication year - 2013
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1065
Subject(s) - mutagenesis , saturated mutagenesis , primer (cosmetics) , computational biology , site directed mutagenesis , protein engineering , directed evolution , biology , genetics , microbiology and biotechnology , chemistry , mutation , biochemistry , enzyme , gene , organic chemistry , mutant
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, M g 2+ , and d NTP s. For this, only nine PCRs need to be performed. The strategy (termed as O pti M ega) was not only successfully applied for the construction of one multiple‐site saturation mutagenesis library of halohydrin dehalogenase H he C , which failed to be constructed previously using the standard Q uik C hange™ protocol, but also expanded the construction of two high‐quality random mutagenesis libraries of H he A and H he C . Most importantly, O pti M ega offers a quick and simple way of constructing random mutagenesis libraries by eliminating the ligation step. Our results demonstrated that the O pti M ega strategy could greatly strengthen the potential of megaprimer PCR mutagenesis for library construction.