Application of FISH for in situ detection and quantification of DNA breakage

We describe a simple procedure that allows the use of fluorescence in situ hybridization (FISH) for in situ detection of DNA strand breaks in single c ells (DBD-FISH: DNA Breakage Detection-FISH), After trapping within an agar ose microgel, cells are incubated in an unwinding alkaline solution, deprot einized and dehydrated. Areas of single-stranded DNA are generated by the a lkaline solution in proportion to the degree of DNA strand breakage. These then act as targets for FISH of whole genomic or region-specific probes (te lomeric, human chromosome 8 painting, human alphoid DXZ1 locus, and human c -erbB-2 cosmid probes). Measurement of the amount and surface of FISH signa ls provides information on the breakage level in probed areas, permitting t he assessment of possible intragenomic differences in sensitivity as well a s intercellular heterogeneity in DNA damage induction or repair.