Rapid and scalable in vitro production of single-stranded DNA

Dionis Minev; Richard Guerra; Jocelyn Y. Kishi; Cory J. Smith; Elisha Krieg; Khaled Said; Amanda Hornick; Hiroshi M. Sasaki; Gabriel T. Filsinger; Brian J. Beliveau; Peng Yin; George McDonald Church; William M. Shih

doi:10.1101/558429

Molecular BiologybioRxivFeb 22, 2019

Preprint

Rapid and scalable in vitro production of single-stranded DNA

Dionis Minev, Richard Guerra iD, Jocelyn Y. Kishi iD, Cory J. Smith iD, Elisha Krieg iD, Khaled Said, Amanda Hornick iD, Hiroshi M. Sasaki iD, Gabriel T. Filsinger iD, Brian J. Beliveau iD, Peng Yin iD, George McDonald Church iD, William M. Shih iD

Abstract

We present a rapid, scalable, user-friendly method for in vitro production of high-purity single-stranded DNA (ssDNA) ranging from 89-3315 nucleotides in length. 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 the recovered ssDNA can be used for CRISPR/Cas9 homology-directed repair in human cells, DNA-origami folding, and fluorescent in situ hybridization.

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