46] Analysis of replication intermediates by two-dimensional agarose gel electrophoresis

Abstract

Publisher Summary This chapter focuses on the analysis of replication intermediates by two-dimensional (2D) agarose gel electrophoresis. The first step in the analysis of replication intermediates on 2-D gels involves the isolation of branched molecules in a way that preserves their structure. Branched molecules are delicate, and shearing is minimized by use of large-bore pipette tips and gentle hand mixing. Shearing can lead to loss of replication intermediates, as well as generation of novel branched artifacts. Exposure of the DNA to excessive heat is avoided in order to preserve the integrity of DNA at the fork. Restriction fragments of larger or smaller size can be analyzed on 2-D gels if some modifications are made to the electrophoresis conditions. Finding the correct electrophoresis conditions for larger or smaller fragments require some experimentation using fragments of known size and replication pattern. Fragments that are larger than 5.5 to 6 kb must be run under conditions of lower agarose concentration and lower voltage in both dimensions in order to separate successfully the bubble and simple Y arcs. If gels are run under the conditions intended for smaller fragments (2 to 5.5 kb), the bubble and simple Y patterns are not clearly separated, and the signals for each are distorted. After the second dimension of electrophoresis, nonreplicating molecules are seen as a shallow arc, whereas the rare replicating molecules run above this arc.