User-defined single pot mutagenesis using unamplified oligo pools

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.

Keywords: deep mutational scanning; directed evolution; nicking mutagenesis; oligo pools; saturation mutagenesis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Gene Library*
  • Mutagenesis, Site-Directed / methods*
  • Oligodeoxyribonucleotides* / chemical synthesis
  • Oligodeoxyribonucleotides* / chemistry
  • Oligodeoxyribonucleotides* / genetics
  • Plasmids / chemistry
  • Plasmids / genetics
  • Protein Engineering / methods*

Substances

  • Oligodeoxyribonucleotides