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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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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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