Reverse transcription and amplification of 2′F DNA by commercially available DNA polymerases

Abstract

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