用户定义的单个小瓶诱变使用未扩增的寡聚池。
User-defined single pot mutagenesis using unamplified oligo pools.
机构信息
Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI, USA.
Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO, USA.
出版信息
Protein Eng Des Sel. 2019 Sep 10;32(1):41-45. doi: 10.1093/protein/gzz013.
Abstract
User-defined mutagenic libraries are fundamental for applied protein engineering workflows. Here we show that unamplified oligo pools can be used to prepare site saturation mutagenesis libraries from plasmid DNA with near-complete coverage of desired mutations and few off-target mutations. We find that oligo pools yield higher quality libraries when compared to individually synthesized degenerate oligos. We also show that multiple libraries can be multiplexed into a single oligo pool, making preparation of multiple libraries less expensive and more convenient. We provide software for automatic oligo pool design that can generate mutagenic oligos for saturating or focused libraries.
摘要
用户定义的诱变文库是应用蛋白质工程工作流程的基础。在这里，我们表明，未扩增的寡核苷酸池可用于从质粒 DNA 制备接近完全覆盖所需突变且突变较少的定点饱和突变文库。我们发现，与单独合成的简并寡核苷酸相比，寡核苷酸池产生的文库质量更高。我们还表明，可以将多个文库多路复用到单个寡核苷酸池中，从而降低了多个文库的制备成本并使其更方便。我们提供了自动寡核苷酸池设计软件，可以为饱和或聚焦文库生成诱变寡核苷酸。
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