The Influence of the Capping Step During Solid-Phase Phosphoramidite Synthesis of Oligonucleotides on Synthetic...

Errors in de novo synthesized DNA can originate from the oligonucleotides used during assembly. Oligonucleotides may contain substitutions, deletions, and insertions resulting from either incomplete reactions at individual steps of the phosphoramidite synthetic cycle or various side reactions. In this study, we quantitatively assessed errors in both gene constructs assembled from synthetic oligonucleotides by Sanger sequencing and in synthetic oligonucleotides by NGS. Our data demonstrate that side reactions involving carboxylic acid anhydrides during the capping step of oligonucleotide synthesis lead to the modification of guanine residues. This guanine modification subsequently results in the accumulation of G to A substitutions in the final gene constructs. We show that the error rate can be reduced by replacing the standard acetic anhydride-based capping mixture with anhydrides of carboxylic acids weaker than acetic acid. Furthermore, a more significant reduction in errors is achievable by using capping reagents based on phosphoramidite chemistry.