MutS-mediated rapid and cost-effective error correction in in vitro DNA synthesis

Abstract

The in vitro synthesis of DNA oligonucleotides and their subsequent assembly into longer target molecules represents a pivotal technique within the field of synthetic biology. However, the occurrence of side reactions and the inherent coupling efficiency of the synthesis process lead to the unavoidable introduction of errors into the resulting DNA. Consequently, there is a pressing demand for a straightforward, cost-effective, and efficient method for error correction. In this study, eleven Cbm3-Egfp-MutS fusion proteins were recombinantly expressed and purified, and their capacity to bind heteroduplex DNA was assessed. Among the MutS proteins, TaMutS and TtMutS exhibited thermal stability and effectively distinguished DNA containing mismatches. Following this, a simple, rapid, efficient, and economical error correction method was devised utilizing a homemade spin column composed of amorphous cellulose and a filter tip. The quantitative affinity of EcMutS, TaMutS, and TtMutS for all conceivable single-base errors was determined, and the efficacy of combining MutS proteins for error correction was evaluated. The error rate in synthesized DNA was reduced by a factor ranging from 2.15--8.17, with the material cost for a single reaction amounting to $0.032. The reaction volume was limited to 10 μL, and the reaction could be completed within 20 minutes.