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

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-ofconcept 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 ``genomein-a-box'' approach to study chromosome organization from the bottom up.

Automated summary

Chromosome structure and function were studied through various cell-based methods and in vitro single-molecule techniques. The researchers developed a method to obtain megabase-pair-length deproteinated DNA for in vitro studies. By isolating chromosomes from bacterial cells and enzymatically digesting native proteins, they removed DNA-binding proteins and observed DNA compaction and motion. The study suggests the feasibility of a "genome-in-a-box" approach to study chromosome organization.