Evolving a High-Performance Terminal Deoxynucleotidyl Transferase for Enzymatic DNA Synthesis
Stephanie M. ForgetiD, Mikayla KrawczykiD, Anders M. KnightiD, Charlene ChingiD, Rachelle A. CopelandiD, Niusha MahmoodiiD, Melissa MayoiD, James NguyeniD, Amanda TaniD, Mathew G. MilleriD, Jonathan A. VroomiD, Stefan LutziD
Abstract
Enzymatic DNA synthesis, using stepwise nucleotide addition catalyzed by template-independent polymerases, promises higher efficiency, quality, and sustainability than todays industry standard phosphoramidite-based processes. We report on the directed evolution of a terminal deoxynucleotidyl transferase that uses 3-phosphate blocked dNTPs to control the polymerization reaction and demonstrates high activity for these modified substrates and improved template promiscuity and thermostability.
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