A PCR-free rapid protocol for one-pot construction of highly diverse genetic libraries. Academic Article uri icon


  • In vitro protein display methods can access extensive libraries (e.g., 1012-1014) and play an increasingly important role in protein engineering. However, the preparation of large libraries remains a laborious and time-consuming process. Here we report an efficient one-pot ligation & elongation (L&E) method for sizeable synthetic library preparation free of PCR amplification or any purification steps. As a proof of concept, we constructed an ankyrin repeat protein templated synthetic library with 1011 variants in 150 L volume. The entire process from the oligos to DNA template ready for transcription is linearly scalable and took merely 90 minutes. We believe this L&E method can significantly simplify the preparation of synthetic libraries and accelerate in vitro protein display experiments.

published proceedings

  • PLoS One

author list (cited authors)

  • Woolley, M., & Chen, Z.

citation count

  • 0

complete list of authors

  • Woolley, Michael||Chen, Zhilei

editor list (cited editors)

  • Fujii, H.

publication date

  • January 2022