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A. S. Thompson, S. E. Barrett, A. G. Weiden, A. Venkatesh, M. K. C. Seto, S. Z. P. Gottlieb, and A. M. Leconte. 2020. Accurate and efficient one-pot reverse transcription and amplification of 2’ fluoro-modified nucleic acids by commercial DNA polymerases. Biochemistry : . Full Article

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) were able to reverse transcribe and amplify synthetic 2'F M-DNA in a single reaction with < 5 x 10^-3 errors 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 conversion of 2'F M-DNA to DNA in less than one hour using commercially available materials, significantly improving the ease of use of M-DNA.

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