title: Electron microscopy can be used to measure DNA supertwisting.
abstract: We have examined the use of electron microscopy (EM) to measure the degree of supertwisting of covalently closed circular fd and pBR322 DNAs. Band counting agarose gel electrophoresis was used to quantitate the number of twists in these DNAs. DNA species having moderate-to-low superhelical densities were prepared by treatment of the DNA with Drosophila topoisomerase 1 in the presence of ethidium bromide (EtBr) followed by isolation of discrete DNA topoisomers from preparative gels. These DNAs were then examined by EM using a direct mounting method employing a buffer containing spermidine. The crossovers in individual DNA molecules were counted and compared to the supertwist values obtained from gel studies. The results show that EM can provide a reliable estimate of the number of supertwists in DNAs having low-to-moderate superhelical densities.
mesh_terms: D004264:DNA Topoisomerases, Type I; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004278:DNA, Superhelical / Q000032:analysis* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D004996:Ethidium; D008854:Microscopy, Electron; D010957:Plasmids
chemical_list: D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004264:DNA Topoisomerases, Type I; D004996:Ethidium