DNA supercoil introduced to directly visualize individual plectonemes along supercoiled DNA which would further allow to study the interactions of DNA processing proteins with supercoiled DNA. In that study, Sytox Orange (an intercalating dye), has been used to induce supercoiling on surface tethered DNA molecules. DNA supercoiling is important for DNA packaging within all cells. Because the length of DNA can be thousands of times that of a cell, packaging this genetic material into the cell or nucleus (in eukaryotes) is a difficult feat. Supercoiling of DNA reduces the space and allows for DNA to be packaged. In prokaryotes, plectonemic supercoils are
Single-cell DNA template strand sequencing cells are treated with bromodeoxyuridine (BrdU) which is then incorporated into their nascent DNA, acting as a substitute for thymidine. After at least one replication event has occurred, the daughter cells are synchronized at the G2 phase and individually separated by fluorescence-activated cell sorting (FACS). The cells are directly sorted into lysis buffer and their DNA is extracted. Having been arrested at a specified number of generations (usually one), the inheritance patterns of sister chromatids can be assessed. The following methods concentrate on the DNA sequencing of a single daughter cell’s DNA. At this point the chromosomes are composed of