A PCR-free rapid protocol for one-pot construction of highly diverse genetic libraries

In vitro protein display methods can access extensive libraries (e.g., 10(12)-10(14)) 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 10(11) variants in 150 mu 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.

Automated summary

This article introduces an efficient one-pot ligation & elongation method for preparing large synthetic libraries without PCR amplification or any purification steps. Through this method, synthetic libraries with a large number of variants can be rapidly prepared in a short time, accelerating the progress of related experiments.