Analysis of meiotic recombination intermediates by two-dimensional gel electrophoresis

Abstract

During meiosis, programmed double strand breaks give rise to crossover and non-crossover recombination products. Meiotic recombination products are formed via several branched intermediates, including single end invasions and double Holliday junctions. Two-dimensional gel electrophoresis provides a sensitive and specific approach for detecting branched recombination intermediates, determining their genetic requirements, and enriching intermediates for further analysis. Here, we describe analysis of branched recombination intermediates in the yeast Saccharomyces cerevisiae by two-dimensional gel electrophoresis. We also provide an introduction to meiotic time-course procedures, stabilization of branched DNA molecules by interstrand crosslinking, extraction of genomic DNA from meiotic cultures, and quantitative analysis of two-dimensional gel blots.