From fragmented workflows to integrated pipelines: Bridging enzymatic DNA synthesis, assembly, and MutS-based error correction

Abstract

The construction of designer DNA sequences at the gene-to-pathway scale is a cornerstone of synthetic biology. In practice, however, oligonucleotide synthesis, fragment assembly, and error correction remain largely disconnected, with limited consideration of how upstream choices constrain downstream outcomes. This fragmentation imposes a ceiling on the fidelity, throughput, and scalability of gene construction, particularly for sequences in the tens-of-kilobases range. Existing automation has been developed separately for the front end, such as oligonucleotide synthesis and short fragment assembly, and for the back end, such as large-fragment concatenation, without bridging them into an error-aware pipeline. In this review, we argue that a truly integrated pipeline cannot be achieved simply by placing existing modules in sequence, but instead requires understanding the dependencies that couple synthesis, assembly, and fidelity control. We first trace how errors arise during oligonucleotide synthesis and propagate through hierarchical assembly, establishing that error correction is not optional but decisive for construction success. We then compare correction strategies and show that MutS-based enzymatic mismatch depletion offers superior correction efficiency and workflow compatibility over alternatives. Building on this, we argue that the emergence of enzymatic DNA synthesis, operating under aqueous conditions compatible with downstream enzymatic processes, creates an opportunity to restructure the gene construction pipeline. This opens the door to integrating oligonucleotide synthesis, assembly, and MutS-based error correction into a continuous automated workflow. We propose that such integration, rather than separate automation of individual stages, is the path toward high-fidelity, high-throughput, scalable construction of gene- and pathway-scale DNA sequences.

Keywords: Automation; Enzymatic DNA synthesis; Error correction; Fragment assembly; High-fidelity DNA synthesis; MutS-based mismatch depletion; Synthetic biology; Workflow integration.