Alpha satellite sequences in extrachromosomal circular DNA from human cell lines. (A) A diagram of 2D gel electrophoretic patterns of genomic DNA generated by populations of linear and circular molecules heterogeneous in size. Each arc consists of molecules sharing the same structure, but differing in mass (Cohen and Lavi 1996). Hybridization with specific probes enables detection of specific sequences within the population of eccDNA. (B-E) Total DNA from human cell lines (indicated in each panel) was cleaved with Eco RI, digested with 'plasmid safe' DNase, analysed on two-dimensional (2-D) gels and hybridized with an 'all centromer' polymerase chain reaction product directed to the conserved region of the different types of alpha satellite. In all cases the probe hybridized with the linear DNA and with the arc that corresponds to open circles. (F-I) DNA from HEK-293 (human embryonic kidney cells) cells was mixed with a 10.8 kb plasmid prior to 2D gel analysis. Ethidium bromide (EtBr) staining of the gel reveals the three forms of the plasmid as well as the arc of linear DNA (I). These forms are visible upon hybridization with a plasmid probe (H). Hybridization with alpha satellite probe reveals the arcs of linear DNA and open circles (F). Note that the arc of open circles has a local deformation at the migration point of the over-loaded relaxed form of the plasmid. This is further confirmed by the merged image of panel F with a shorter exposure of panel H (G). White arrows indicate the plasmid forms; R = relaxed; SC = supercoiled.