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Ramified rolling circle amplification for efficient and flexible synthesis of nucleosomal DNA sequences

View ORCID ProfileClara L. van Emmerik, Ivana Gachulincova, Vincenzo R. Lobbia, Mark A. Daniëls, Hans A. Heus, Abdenour Soufi, Frank H.T. Nelissen, View ORCID ProfileHugo van Ingen
doi: https://doi.org/10.1101/676528
Clara L. van Emmerik
From the Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands
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  • ORCID record for Clara L. van Emmerik
Ivana Gachulincova
MRC Centre for Regenerative Medicine, University of Edinburgh, United Kingdom
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Vincenzo R. Lobbia
From the Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands
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Mark A. Daniëls
From the Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands
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Hans A. Heus
Institute for Molecules and Materials, Radboud University Nijmegen, The Netherlands
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Abdenour Soufi
MRC Centre for Regenerative Medicine, University of Edinburgh, United Kingdom
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Frank H.T. Nelissen
Institute for Molecules and Materials, Radboud University Nijmegen, The Netherlands
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Hugo van Ingen
From the Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands
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  • ORCID record for Hugo van Ingen
  • For correspondence: h.vaningen@uu.nl
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ABSTRACT

Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that using ramified rolling circle amplification (RCA) milligram amounts of a genomic nucleosomal DNA fragment can be produced in a scalable, one-pot reaction overnight. The ramified RCA reaction is more efficient than the existing methods, is flexible in DNA sequence and shows a 10-fold increase in yield compared to PCR, rivalling the production using plasmids. We demonstrate the method by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.

Footnotes

  • - minor textual updates to clarify the general applicability of the method - minor editing of figures

  • The abbreviations used are

    rRCA
    ramified rolling circle amplification;
    PCR
    polymerase chain reaction;
    dNTPs
    deoxyribose nucleoside triphosphates
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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    Posted July 18, 2019.
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    Ramified rolling circle amplification for efficient and flexible synthesis of nucleosomal DNA sequences
    Clara L. van Emmerik, Ivana Gachulincova, Vincenzo R. Lobbia, Mark A. Daniëls, Hans A. Heus, Abdenour Soufi, Frank H.T. Nelissen, Hugo van Ingen
    bioRxiv 676528; doi: https://doi.org/10.1101/676528
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    Ramified rolling circle amplification for efficient and flexible synthesis of nucleosomal DNA sequences
    Clara L. van Emmerik, Ivana Gachulincova, Vincenzo R. Lobbia, Mark A. Daniëls, Hans A. Heus, Abdenour Soufi, Frank H.T. Nelissen, Hugo van Ingen
    bioRxiv 676528; doi: https://doi.org/10.1101/676528

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