Fast-painting of human metaphase spreads using a chromosome-specific, repeat-depleted DNA library probe.
For chromosome painting, 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 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 q-arm of chromosome 15 (Fast-Painting) for human lymphocyte metaphase spreads. Following painting without Cot-I 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.
Durm
M
M
University of Heidelberg, Germany.
Schüssler
L
L
Münch
H
H
Craig
J
J
Ludwig
H
H
Hausmann
M
M
Cremer
C
C
D000328:Adult; D002884:Chromosomes, Human, Pair 15 / Q000737:chemistry*; D015723:Gene Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008214:Lymphocytes / Q000737:chemistry; D008677:Metaphase / Q000235:genetics*; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D015345:Oligonucleotide Probes; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted; D013194:Staining and Labeling / Q000379:methods
D015345:Oligonucleotide Probes