Manufacturing DNA in E. coli yields higher fidelity DNA thanin vitroenzymatic synthesis

Abstract

AbstractThe rise of biotechnologies such as gene therapy have brought DNA vectors to the forefront of pharmaceutical development. The quality of the genetic starting material plays a pivotal role in determining the quality of the final product. In this study we examined the fidelity of DNA replication using enzymatic methods (in vitro) compared to plasmid DNA producedin vivoinE. coli. Next-generation sequencing approaches predominantly rely onin vitropolymerases, 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 toxicsacBgene. Our findings show that DNA production inE. coliresults in significantly fewer LOF mutations (approximately 80-to 3000-fold less) compared to various enzymatic DNA synthesis methods. This includes the most accurate PCR polymerase (Q5) and a commonly employed rolling circle amplification (RCA) DNA polymerase (Phi29). These results suggest that using low-fidelity starting material DNA synthesizedin vitroby PCR or RCA may introduce a substantial number of impurities, potentially affecting the quality and yield of final pharmaceutical products. In summary, our study underscores that DNA synthesizedin vitrohas a significantly higher mutation rate than DNA produced traditionally inE. coli. Therefore, utilizingin vitroenzymatically-produced DNA in biotechnology and biomanufacturing may entail considerable fidelity-related risks, while DNA starting material derived fromE. colisubstantially mitigates this risk, enhancing overall quality in the production processes.