FAST-PAINTING OF HUMAN METAPHASE SPREADS USING A CHROMOSOME-SPECIFIC,REPEAT-DEPLETED DNA LIBRARY PROBE

For chromosome painting, in situ suppression of repetitive DNA sequenc es has been well established. Such standard protocols usually require large amounts of cot-l DNA(R). Recently, it has become possible to dep lete repetitive DNA sequences from library probes by magnetic purifica tion and PCR-assisted affinity chromatography. These ''repeat-depleted library probes'' appear to be extremely useful for Fast-FISH, a techn ique that omits denaturing chemical agents such as formamide in the hy bridization buffer, resulting in a substantial acceleration and simpli fication of the complete protocol. Shown here is the application of Fa st-FISH to a repeat-depleted, directly fluorochrome-labeled library pr obe of the q-arm of chromosome 15 (Fast-Painting) for human lymphocyte metaphase spreads. Following painting without Cot-l DNA and without f ormamide, visual inspection revealed sufficient chromosome painting af ter a few hours of hybridization. The fluorescence signals of the labe ling sites were analyzed after hybridization times of I and 2 h (in on e case, 4 h) using digital fluorescence microscopy. The painting effic iency expressed in values of relative fluorescence signal ratios was q uantitatively evaluated by image analysis using line-scan procedures a nd area-morphometry of mean luminance. Two preparation protocols (etha nol dehydration without and with RNase A treatment followed by pepsin digestion for Sour different exposure times) were compared. These resu lts indicated that RNase ii treatment and pepsin digestion are steps t hat can be omitted.