Print Email Facebook Twitter Extracting and characterizing protein-free megabase-pair DNA for in vitro experiments Title Extracting and characterizing protein-free megabase-pair DNA for in vitro experiments Author Holub, M. (TU Delft BN/Cees Dekker Lab; Kavli institute of nanoscience Delft) Birnie, A.T.F. (TU Delft BN/Cees Dekker Lab; Kavli institute of nanoscience Delft) Japaridze, A. (TU Delft Dynamics of Micro and Nano Systems; Kavli institute of nanoscience Delft) van der Torre, J. (TU Delft BN/Cees Dekker Lab; Kavli institute of nanoscience Delft) Ridder, Maxime den (Student TU Delft) de Ram, C. (TU Delft BT/Environmental Biotechnology) Pabst, Martin (TU Delft BT/Environmental Biotechnology) Dekker, C. (TU Delft BN/Cees Dekker Lab; Kavli institute of nanoscience Delft) Date 2022 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. Subject bottom-up biologychromosome organizationCP: Molecular biologyDNADNA-binding proteinsfluorescence imaginggenomemass spectrometry To reference this document use: http://resolver.tudelft.nl/uuid:5f83a2d2-6109-4143-8cfc-469155a65fe9 DOI https://doi.org/10.1016/j.crmeth.2022.100366 Source Cell Reports Methods, 2 (12) Part of collection Institutional Repository Document type journal article Rights © 2022 M. Holub, A.T.F. Birnie, A. Japaridze, J. van der Torre, Maxime den Ridder, C. de Ram, Martin Pabst, C. Dekker Files PDF 1_s2.0_S2667237522002685_main.pdf 2.86 MB Close viewer /islandora/object/uuid:5f83a2d2-6109-4143-8cfc-469155a65fe9/datastream/OBJ/view