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

ABSTRACT: 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 (in vitro) compared to plasmid DNA produced in vivo in E. coli. Next-generation sequencing approaches rely on in vitro 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 sacB gene. Our findings show that DNA production in E. coli results in significantly fewer LOF mutations (80- to 3,000-fold less) compared to enzymatic DNA replication methods such as polyme

SUBMITTER: Hersch SJ 

PROVIDER: S-EPMC10951457 | biostudies-literature | 2024 Jun

REPOSITORIES: biostudies-literature