Gel Electrophoresis Assays for Analyzing DNA Double-Strand Breaks in Saccharomyces cerevisiae at Various Spatial Resolutions

Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Methods in Molecular Biology (Impact Factor: 1.29). 01/2009; 557:117-42. DOI: 10.1007/978-1-59745-527-5_9
Source: PubMed


Meiotic recombination is triggered by programmed DNA double-strand breaks (DSBs), which are catalyzed by Spo11 protein in a type II topoisomerase-like manner. Meiotic DSBs can be detected directly using physical assays (gel electrophoresis, Southern blotting, and indirect end-labeling) applied to samples of genomic DNA from sporulating cultures of budding and fission yeast. Such assays are extremely useful for quantifying and characterizing many aspects of the initiation of meiotic recombination, including the timing of DSB formation relative to other events, the distribution of DSBs across the genome, and the influence on DSB formation of mutations in recombination factors and other gene products. By varying the type of gel electrophoresis and other parameters, the spatial resolution of DSB analysis can range from single nucleotides up to whole yeast chromosomes.

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Available from: Hajime Murakami, Sep 25, 2015
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    • "Cultures of sae2D strains were grown in liquid YPA (1% yeast extract, 2% Bacto peptone, 1% potassium acetate) for 13.5 hr at 30°, harvested, resuspended in 2% potassium acetate, and incubated at 30°. Samples were collected at 0 and 8 hr, and DNA was prepared for conventional agarose electrophoresis (Murakami et al. 2009). DNA was digested with BglII and probed with part of the YHR018c open reading frame (SGD coordinates 140353–140717). "
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