Analysis and control of untemplated DNA polymerase activity for guided synthesis of kilobase-scale DNA sequences

DNA polymerases are complex molecular machines able to replicate genetic material using a template-driven process. While the copying function of these enzymes is well established, their ability to perform untemplated DNA synthesis is less well characterized. Here, we explore the ability of DNA polymerases to synthesize DNA fragments in the absence of template. We use long-read nanopore sequencing and real-time PCR to observe the synthesis of pools of DNA products derived from a diverse set of natural and engineered DNA polymerases across varying temperatures and buffer compositions. We detail the features of the DNA fragments generated, enrichment of select sequence motifs, and demonstrate that the sequence composition of the synthesized DNA may be altered by modifying environmental conditions. This work provides an extensive data set to better discern the process of untemplated DNA polymerase activity and may support its potential repurposing as a technology for the guided synthesis of DNA sequences on the kilobase-scale and beyond.