Fast-Painting of Human Metaphase Spreads Using a Chromosome-Specific, Repeat-Depleted DNA Library Probe. (May 1998) - Durm, M.
For chromosome painting, in situ suppression of repetitive DNA sequences has been well established. Such standard protocols usually require large amounts of Cot-1 DNA ® . Recently, it has become possible to deplete repetitive DNA sequences from library probes by magnetic purification and PCR-assisted affinity chromatography. These "repeat-depleted library probes" appear to be extremely useful for Fast -FISH, a technique that omits denaturing chemical agents such as formamide in the hybridization buffer, resulting in a substantial acceleration and simplification of the complete protocol. Shown here is the application of Fast -FISH to a repeat-depleted, directly fluorochrome-labeled library probe of the qarm of chromosome 15 ( Fast -Painting) for human lymphocyte metaphase spreads. Following painting without Cot-1 DNA and without formamide, visual inspection revealed sufficient chromosome painting after a few hours of hybridization. The fluorescence signals of the labeling sites were analyzed after hybridization times of 1 and 2 h (in one case, 4 h) using digital fluorescence microscopy. The painting efficiency expressed in values of relative fluorescence signal ratios was quantitatively evaluated by image analysis using line-scan procedures and area-morphometry of mean luminance. Two preparation protocols (ethanol dehydration without and with RNase A treatment followed by pepsin digestion for four different exposure times) were compared. These results indicated that RNase A treatment and pepsin digestion are steps that can be omitted.