Fast-painting of XXXX metaphase spreads using a chromosome-specific, repeat-depleted DNA library probe.

@entity1 @entity27390 @entity2008 @entity30555 @entity1068
For @entity2008 , in situ suppression of repetitive DNA sequences has been well established. Such standard protocols usually require large amounts of Cot-I 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 @entity27390 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 q-arm of chromosome 15 (Fast-Painting) for @entity1 lymphocyte metaphase spreads. Following painting without Cot-I DNA and without @entity27390 , visual inspection revealed sufficient @entity2008 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 ( @entity1068 dehydration without and with @entity30555 treatment followed by pepsin digestion for four different exposure times) were compared. These results indicated that @entity30555 treatment and pepsin digestion are steps that can be omitted.
