DNA is a foundational tool in biotechnology and synthetic biology but is limited by sensitivity to DNA-modifying enzymes. Recently, researchers have identified DNA polymerases that can enzymatically synthesize long oligonucleotides of modified DNA (M-DNA) that are resistant to DNA-modifying enzymes. Most applications require M-DNA to be reverse transcribed, typically using a RNA reverse transcriptase, back into natural DNA for sequence analysis or further manipulation. Here, we tested commercially available DNA-dependent DNA polymerases for their ability to reverse transcribe and amplify M-DNA in a one-pot reaction. Three of the six polymerases chosen (Phusion, Q5, and Deep Vent) could reverse transcribe and amplify synthetic 2′F M-DNA in a single reaction with <5 × 10–3 error per base pair. We further used Q5 DNA polymerase to reverse transcribe and amplify M-DNA synthesized by two candidate M-DNA polymerases (SFP1 and SFM4–6), allowing for quantification of the frequency, types, and locations of errors made during M-DNA synthesis. From these studies, we identify SFP1 as one of the most accurate M-DNA polymerases identified to date. Collectively, these studies establish a simple, robust method for the conversion of 2′F M-DNA to DNA in <1 h using commercially available materials, significantly improving the ease of use of M-DNA.

Modified‐DNA polymerases have been evolved that can synthesize long strands of modified oligonucleotides. The resulting modified DNA (M‐DNA) has many high‐value potential applications, such as clinical diagnostics and therapeutics. To fully apply M‐DNA, it must be reverse transcribed back into natural DNA. Previously, error prone reverse transcriptases have been required for this step, and then the resulting DNA has to be amplified in a separate amplification reaction. Using a synthetic template with 2′F modified nucleotides, we tested a panel of commercially available DNA‐dependent DNA polymerases for their ability to reverse transcribe (RT) and amplify M‐DNA in a one‐pot reaction. We found that four of the six polymerases chosen (Phusion, Q5, Deep Vent, and Vent) were able to RT and amplify synthetic 2′F modified DNA in a single reaction. These products were sent in for high‐throughput sequencing and it was determined that Q5, Deep Vent and Phusion had between 90 and 95% matched reads, while Vent had less than 85%. The conditions of this process (reaction buffer and PCR cycling conditions) were also shown to affect the accuracy, but to a lesser degree. These polymerases were also able to RT and amplify enzymatically synthesized 2′F templates, allowing us to use this as a tool to quantitatively determine error rates of M‐DNA polymerases using high‐throughput sequencing. This demonstrated that P1, one of our labs best modified‐DNA polymerases, was almost four times more accurate than SFM4‐6, the fields previous best enzyme. These discoveries will enable the wider use of 2′F M‐DNA, as well as add to the. evidence supporting the use of commercially available DNA polymerases for reverse transcription reactions. Support or Funding Information National Science Foundation