Extracting and characterizing protein-free megabase-pair DNA for in vitro experiments
Martin Holub (Kavli institute of nanoscience Delft, TU Delft - BN/Cees Dekker Lab)
Anthony Birnie (TU Delft - BN/Cees Dekker Lab, Kavli institute of nanoscience Delft)
Aleksandre Japaridze (Kavli institute of nanoscience Delft, TU Delft - Dynamics of Micro and Nano Systems)
Jaco van der Torre (Kavli institute of nanoscience Delft, TU Delft - BN/Cees Dekker Lab)
Maxime den Ridder (Student TU Delft)
Carol de Ram (TU Delft - BT/Environmental Biotechnology)
Martin Pabst (TU Delft - BT/Environmental Biotechnology)
Cees Dekker (Kavli institute of nanoscience Delft, TU Delft - BN/Cees Dekker Lab)
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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.
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