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

• DOI: 10.1021/acs.biochem.0c00494
• Publication Date: 2020-07-13T21:03:03Z
• Authors: Arianna S. Thompson; Susanna E. Barrett; Aurora G. Weiden; Ananya Venkatesh; Madison K. C. Seto; Simone Z. P. Gott...; Aaron M. Leconte
• Keywords: DNA clamp; Primase; DNA polymerase II; Sequencing by ligation; In vitro recombination

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