Two-dimensional gel electrophoresis allows direct detection of DNA replication and recombination intermediates in preparations of total genomic DNA. This technique is widely used to identify replication origins in the yeast genome and is based on the different mobility in agarose gels of linear and branched DNA molecules depending on their mass and structure. During the first dimension, low-voltage and a low-percentage agarose gel favors separation of the molecules by their mass and minimizes the effect of their structure. In contrast, during the second dimension, a higher voltage, a higher percentage agarose gel, and the presence of ethidium bromide significantly delays the migration of branched structures relative to linear molecules of the same mass. This technique is appropriate for the detection of replication initiation and, therefore, an active origin of replication, within regions of approximately 2.5-5.5 kb. Consequently, it is not well suited for genome-wide replication analyses.