Supporting information for Zhou et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.212392399
Fig. 4.
The principle of neutral/alkaline two-dimensional (N/A 2D) gel analysis is schematically shown. The first dimension of the agarose gel is run in neutral conditions in which restriction-digested DNA molecules enriched in replication intermediates are separated according to their mass. The second dimension is run in alkaline conditions in order to separate parental and nascent strands. The blot is probed with unique end fragments (solid rectangles). Signals detected by autoradiography (a–e) are schematically shown. The 1N spot (dot a) represents linear molecules of the same size as the restriction fragment. The horizontal line (b) represents the parental strands of the replication intermediates, which run at the same position in the second dimension because they have the same molecular weight. The diagonal line (c) emanating from the parental strands represents nascent strands of different sizes, ranging from very small to as large as the parental strands. If replication forks move from 3' to 5' as the schematic shows, a probe located at the 3' end of a fragment will detect nascent strands of sizes ranging from small to parental-sized, while probes located at the 5' end will detect nascent strands only of a relatively large size. If forks move in both directions through a segment, then nascent strands of multiple sizes will be detected with probes for both 5' and 3' ends of this segment. (d) The cross hybridization signal to the nonreplicating linear molecules. (e) Single-stranded molecules that result from breaks in the linear nonreplicating molecules (from the 1N spot).