Fig. 3. Resolution of junctional molecules. 2DNAGE of AccI-digested human heart DNA samples (not S1-digested), probed for the 4.8 kb OL-containing fragment, after various treatments. (A) Samples h1 and h2 with or without treatment of the first-dimension gel slab to promote branch migration. Note the large increase in material from the X-spike now migrating in the second dimension with the mobility of the unit fragment (arrow). (B) Sample h2 (2 µg) incubated for 20 h in agarose gel plug with or without RuvC. Note the increase in heterogeneous double-stranded DNA digestion products running below the unit fragment, after RuvC treatment. (C) Sample h1 (2 µg) incubated for 1 h with or without 1 U RNase H. (D) Samples h1 and h2 treated with topoisomerase I prior to electrophoresis. (E) Validation of topoisomerase assay. Supercoiled plasmid DNA (500 ng) incubated either with (lane 1) or without (lane 2) 3 U topoisomerase I, or with 15 U of the enzyme in the presence of AccI-digested heart DNA as shown. Lanes marked ++ were incubated with an additional 6 U of the enzyme for a further 2 h, after overnight reactions. The selected conditions used for the main experiment completely remove supercoiling of the test plasmid, and hence should also separate any catenated molecules.