Fig. 3: Experimental evidence of minicircles.

a Southern hybridization (n = 1) results using the cox3 minicircle as probe against R. marinus gDNA. Lane 1: Use of the SpeI restriction enzyme showed a band that corresponds to the size of the assembled cox3 minicircle (linearized DNA; indicated by arrow). Lane 2: Use of BamHI did not result in a single band. However, there was a shift in intensity from supercoiled DNA (lane 2) to linear DNA (lane 3). Lane 3: When minicircles are not treated with a restriction enzyme, three types of DNA conformation (nicked, linear, and supercoiled) are found. Also see Supplementary Fig. 6 and Supplementary Note 2. Lane L: ladder. b FM images (n = 1) of stained LMW DNA with or without restriction enzyme treatment. Undigested LMW DNA molecules appear as dots (right), whereas digested LMW DNA molecules appear as lines (left). c Representative SEM images of LMW DNA with (n = 12) or without (n = 15) restriction enzyme treatment. A large number of supercoiled and open circular DNA molecules (indicated by arrows) are present when LMW DNA is not digested with a restriction enzyme (right), whereas linear DNA molecules are mostly found when LMW DNA is treated with a restriction enzyme (left). d Additional images of supercoiled or open circular LMW DNA molecules from SEM images (n = 12). Measured molecule lengths generally agree with the calculated minicircle length (see Supplementary Note 3). All scale bars indicate 1000 nm.