Rapid and scalable in vitro production of single-stranded DNA

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

doi:10.1101/558429

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Rapid and scalable in vitro production of single-stranded DNA

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

doi: https://doi.org/10.1101/558429

Dionis Minev

1John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

3Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

4Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

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Richard Guerra

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

3Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

4Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

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Jocelyn Y. Kishi

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

6Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

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ORCID record for Jocelyn Y. Kishi

Cory Smith

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

5Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

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Elisha Krieg

7Leibniz Institute for Polymer Research Dresden e.V, Dresden, Germany

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Khaled Said

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

5Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

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Amanda Hornick

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

5Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

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Hiroshi M. Sasaki

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

6Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

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Gabriel Filsinger

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

5Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

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Brian J. Beliveau

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

6Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

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Peng Yin

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

6Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

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George M. Church

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

5Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

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William M. Shih

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA

3Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

4Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

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For correspondence: William.Shih@wyss.harvard.edu

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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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Posted February 22, 2019.

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Rapid and scalable in vitro production of single-stranded DNA

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

bioRxiv 558429; doi: https://doi.org/10.1101/558429

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