Extracting and characterizing protein-free megabase-pair DNA for in vitro experiments

Martin Holub, Anthony Birnie, Aleksandre Japaridze, Jaco van der Torre, Maxime den Ridder, Carol de Ram, Martin Pabst, Cees Dekker*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Chromosome structure and function is studied using various cell-based methods as well as with a range of in vitro single-molecule techniques on short DNA substrates. Here, we present a method to obtain megabase-pair-length deproteinated DNA for in vitro studies. We isolated chromosomes from bacterial cells and enzymatically digested the native proteins. Mass spectrometry indicated that 97%–100% of DNA-binding proteins are removed from the sample. Fluorescence microscopy analysis showed an increase in the radius of gyration of the DNA polymers, while the DNA length remained megabase-pair sized. In proof-of-concept experiments using these deproteinated long DNA molecules, we observed DNA compaction upon adding the DNA-binding protein Fis or PEG crowding agents and showed that it is possible to track the motion of a fluorescently labeled DNA locus. These results indicate the practical feasibility of a “genome-in-a-box” approach to study chromosome organization from the bottom up.

Original languageEnglish
Article number100366
Number of pages17
JournalCell Reports Methods
Volume2
Issue number12
DOIs
Publication statusPublished - 2022

Keywords

  • bottom-up biology
  • chromosome organization
  • CP: Molecular biology
  • DNA
  • DNA-binding proteins
  • fluorescence imaging
  • genome
  • mass spectrometry

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