Manufacturing DNA in E. coli yields higher fidelity DNA than in vitro enzymatic synthesis

Biotechnologies such as gene therapy have brought DNA vectors to the forefront of pharmaceuticals. The quality of starting material plays a pivotal role in determining final product quality. Here we examined the fidelity of DNA replication using enzymatic methods () compared to plasmid DNA produced in . Next-generation sequencing approaches rely on polymerases, which have inherent limitations in sensitivity. To address this challenge, we introduce a novel assay based on loss-of-function (LOF) mutations in the conditionally toxic gene. Our findings show that DNA production in results in significantly fewer LOF mutations (80- to 3000-fold less) compared to enzymatic DNA replication methods such as PCR and rolling circle amplification (RCA). These results suggest that using DNA produced by PCR or RCA may introduce a substantial number of mutation impurities, potentially affecting the quality and yield of final pharmaceutical products. Our study underscores that DNA synthesized has a significantly higher mutation rate than DNA produced traditionally in . Therefore, utilizing enzymatically-produced DNA in biotechnology and biomanufacturing may entail considerable fidelity-related risks, while using DNA starting material derived from substantially mitigates this risk.