High frequency of constitutive alkali-labile sites in mouse major satellite DNA, detected by DNA breakage detection-fluorescence in situ hybridization

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a n ew procedure for detecting and quantifying DNA breaks and alkali-labile sit es in single cells. Cells trapped within an agarose matrix are deproteinize d and treated with an alkaline unwinding solution that transforms DNA break s and alkali-labile sites into single-strand DNA (ssDNA) motifs starting fr om the end of the break. These ssDNA motifs are susceptible to being hybrid ized with whole genome or specific DNA probes, and detected using current F ISH procedures. As DNA breaks increase in a target region, more ssDNA is pr oduced and more DNA probe hybridizes, thus increasing the FISH signal, whic h may be captured and analyzed using a digital image analysis system. This increase can be reflected in the surface area, mean and whole fluorescence intensity of the signal. When intact mouse splenocytes were processed with this technique using a whole genome probe, a very strong background signal was evident when compared with human blood leukocytes. In fact, when using 0.03 M NaOH as the alkaline unwinding solution at 22 degreesC for 2.5 min, the whole fluorescence intensity from mice cells was 50 times higher than t hat from human cells, thus suggesting the existence of a high frequency of constitutive alkali-labile sites in the DNA from mouse cells. Furthermore, when alkaline unwound mouse cells were simultaneously hybridized with the w hole genome probe (FITC-revealed, green) and a major satellite DNA probe (C y-3-labeled, red) both signals appeared co-localized. This result demonstra tes that the high frequency of constitutive alkali-labile sites detected in the mouse genome is mainly located in the major satellite DNA sequences, r esembling the findings from human 5 bp classical satellite DNA sequences. ( C) 2001 Elsevier Science B.V. All rights reserved.