Accurate and Efficient One-Pot Reverse Transcription and Amplification of 2'-Fluoro-Modified Nucleic Acids by Commercial DNA Polymerases

Biochemistry. 2020 Aug 11;59(31):2833-2841. doi: 10.1021/acs.biochem.0c00494. Epub 2020 Jul 27.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA / chemistry*
  • DNA / genetics*
  • DNA / metabolism
  • DNA-Directed DNA Polymerase / metabolism*
  • Halogenation*
  • Models, Molecular
  • Nucleic Acid Amplification Techniques*
  • Nucleic Acid Conformation
  • Reverse Transcription*

Substances

  • DNA
  • DNA-Directed DNA Polymerase