KOD DNA Polymerase Directed Evolution - Optimization of selection parameters

Libraries of Exo- KOD DNA Polymerase (D141A/E143A), a 2-point and a 5-point saturation mutagenesis libraries, were generated and subjected to directed evolution for DNA synthesis or 2'F-dATP incorporation. The 2-point library targeted D404 (catalytic aspartate) and neighboring L403 residues for saturation mutagenesis. The 5-point library targeted D404, L403, F405, L408 and Y409 for saturation mutagenesis. The 2-point library was independently selected with 5 different combinations of selection parameters. Selection products were uniquely barcoded at the 5'-end according to the corresponding selection parameters (Selection 2 (ATG), 5 (TGA), 7 (GAT), 8 (ACT), 11 (CAG)) and pooled together prior to sequencing. The 5-point library was selected with 3 different combinations of selection parameters (Selection 4, 8 and 20).