Modified DNA (M-DNA) has many high-value potential applications, such as in clinical diagnostics and therapeutics. However, 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 had 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 (Q5, Phusion, Deep Vent, and Vent) were able to RT and amplify synthetic 2’F modified DNA, and we optimized this process with respect to Mg2+ concentration and cycling conditions. From these experiments, we determined that Q5, Phusion, and Deep Vent were able to reverse transcribe and amplify 2’F M-DNA with less than 5x10-3 errors per base pair. These polymerases were also able to RT and amplify enzymatically synthesized 2’F templates, allowing us to use this method as a tool to quantitatively determine error rates of M-DNA polymerases SFP1 and SFM4-6 using high-throughput sequencing. We identified SFP1 as one of the most accurate M-DNA polymerases, with an error rate of less than 10x10-3 errors per base pair under all conditions tested, in addition to identifying the types and locations of these errors. Overall, we have developed a simple and efficient method to convert 2’F M-DNA back into DNA in less than an hour using commercially available polymerases, increasing the utility of M-DNA.

Reverse transcription and amplification of 2’F M-DNA in a one-pot reaction by commercially available DNA-dependent DNA polymerases

ACS Spring Meeting 2021

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