Isolation of Meiotic Recombinants from Mouse Sperm
Homologous recombination during meiosis is critical for the formation of gametes. Recombination is initiated by programmed DNA double-strand breaks which preferentially occur at hotspots dispersed throughout the genome. These double-strand breaks are repaired from the homolog, resulting in either a crossover or noncrossover product. Multiple noncrossover events are required for homolog pairing, and at least one crossover is critical for proper chromosome segregation at the first meiotic division. Consequently, homologous recombination in meiosis occurs at high frequencies. This chapter describes how to characterize crossovers and noncrossovers at a hotspot in mice using allele-specific PCR. Amplification of recombinant products directly from sperm DNA is a powerful approach to determine recombination frequencies and map recombination breakpoints, providing insight into homologous recombination mechanisms.
Key wordsMeiotic recombination sperm crossover noncrossover hotspot allele-specific PCR F1 hybrid mice gene conversion homolog
We thank members of the Jasin Laboratory, especially Erika Brunet, and Scott Keeney and members of his laboratory, especially Liisa Kauppi and Esther de Boer, for comments on the manuscript and suggestions on the techniques described in this chapter. This work was supported by a Ruth L. Kirschstein National Research Service Award F32HD51392 (F.C.) and National Institutes of Health Grant RO1HD40916 (M.J.).
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