Title Rapid in vitro production of single-stranded DNA
Date 16.12.2019
Number 57674
Abstract There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ~40–50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 µl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.
Publisher Nucleic Acids Research
Citation Nucleic Acids Research 47 (2019) 11956-11962
Authors Minev, D. ; Guerra, R. ; Kishi, J.Y. ; Smith, C. ; Krieg, E. ; Said, K. ; Hornick, A. ; Sasaki, H.M. ; Filsinger, G. ; Beliveau , B.J. ; Yin, P. ; Church, G.M. ; Shih, W.M.

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