Abstract

DNA breakage detection fluorescence in situ hybridization (DBD-FISH) is an open procedure to detect and quantify DNA breaks in single cells, not only in the whole genome but also on different specific DNA sequence areas. Cells embedded within an inert agarose matrix on a slide are lysed and the resultant nucleoids exposed to a controlled denaturation step. As a consequence, DNA breaks are transformed into restricted single-stranded DNA (ssDNA) motifs, initiated from the ends of the DNA breaks, that may be detected by hybridization with whole genome or specific fluorescent DNA probes. The specific DNA probe selects the chromatin area to be analyzed. As DNA breaks increase within a specific target, and more single-stranded DNA is generated, more probe hybridizes, producing increasing levels of fluorescence. Single-strand breaks (SSBs) and double-strand breaks (DSBs) induced by ionizing radiation, may also be simultaneously discriminated in the same cell if a neutral electrophoresis is performed before denaturation. In this case, the length of migration of the DBD-FISH signal may be proportional to the DSBs, whereas its fluorescence intensity is related to the SSBs. This assay is of particular interest to determine possible intragenomic and intercellular heterogeneity in DNA damage induction and repair. Moreover, this procedure may reveal particular structural features of chromatin structure, like the relative abundance of constitutive alkali-labile sites within a specific DNA sequence. Its application to human sperm cells demonstrates that sperm nuclei containing fragmented DNA do not produce halos of chromatin spreading, or are of very small size, after sequential incubation in acid and lysing solutions. This is the basis of the sperm chromatin dispersion (SCD) test, a very simple, fast, reproducible, and cheapprocedure for the determination of sperm cells with fragmented DNA, which could make the analysis of this possible new parameter of sperm quality feasible even in a basic laboratory.

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