Assessment of enzymatically synthesized DNA for gene assembly

Brooke L Simmons; Nathan D McDonald; Natalie G Robinett

doi:10.3389/fbioe.2023.1208784

Assessment of enzymatically synthesized DNA for gene assembly

Front Bioeng Biotechnol. 2023 Jul 5:11:1208784. doi: 10.3389/fbioe.2023.1208784. eCollection 2023.

Authors

Brooke L Simmons^{1

2}, Nathan D McDonald¹, Natalie G Robinett^{1

3}

Affiliations

¹ U.S. Army Combat Capabilities Development Command (DEVCOM) Chemical Biological Center, Gunpowder, MD, United States.
² Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN, United States.
³ Excet, Inc., Springfield, VA, United States.

Abstract

Phosphoramidite chemical DNA synthesis technology is utilized for creating de novo ssDNA building blocks and is widely used by commercial vendors. Recent advances in enzymatic DNA synthesis (EDS), including engineered enzymes and reversibly terminated nucleotides, bring EDS technology into competition with traditional chemical methods. In this short study, we evaluate oligos produced using a benchtop EDS instrument alongside chemically produced commercial oligonucleotides to assemble a synthetic gene encoding green fluorescent protein (GFP). While enzymatic synthesis produced lower concentrations of individual oligonucleotides, these were available in half the time of commercially produced oligonucleotides and were sufficient to assemble functional GFP sequences without producing hazardous organic chemical waste.

Keywords: benchtop DNA synthesis; chemical DNA synthesis; de novo DNA; enzymatic DNA synthesis; gene assembly.

Grants and funding

This work was funded by the Defense Threat Reduction Agency Joint Science and Technology Office (DTRA-JSTO). The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of Defense or the U.S. government.