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- Title
Exploring the potential of megaprimer PCR in conjunction with orthogonal array design for mutagenesis library construction.
- Authors
Tang, Lixia; Zheng, Kai; Liu, Yu; Zheng, Huayu; Wang, Hu; Song, Chunlei; Zhou, Hong
- 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, Mg2+, and d NTPs. For this, only nine PCRs need to be performed. The strategy (termed as Opti Mega) was not only successfully applied for the construction of one multiple-site saturation mutagenesis library of halohydrin dehalogenase Hhe C, which failed to be constructed previously using the standard Quik Change™ protocol, but also expanded the construction of two high-quality random mutagenesis libraries of Hhe A and Hhe C. Most importantly, Opti Mega offers a quick and simple way of constructing random mutagenesis libraries by eliminating the ligation step. Our results demonstrated that the Opti Mega strategy could greatly strengthen the potential of megaprimer PCR mutagenesis for library construction.
- Subjects
POLYMERASE chain reaction; ORTHOGONAL arrays; MUTAGENESIS; GENE amplification; DEHALOGENASES; LIFE sciences; METHODOLOGY; EVOLUTIONARY theories
- Publication
Biotechnology & Applied Biochemistry, 2013, Vol 60, Issue 2, p190
- ISSN
0885-4513
- Publication type
Article
- DOI
10.1002/bab.1065