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-1 DNA®. Recently, it has become possible to deplete repetitive DNA sequences from library probes by magnetic purification and PCR-assisted...
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| Main Authors: | , , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
1998
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| In: |
BioTechniques
Year: 1998, Volume: 24, Issue: 5, Pages: 820-825 |
| ISSN: | 1940-9818 |
| DOI: | 10.2144/98245dt02 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.2144/98245dt02 Verlag, lizenzpflichtig, Volltext: https://www.future-science.com/doi/10.2144/98245dt02 
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| Author Notes: | M. Durm, L. Schüssler, H. Münch, J. Craig, H. Ludwig, M. Hausmann, C. Cremer |
| Summary: | 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. |
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| Item Description: | Elektronische Reproduktion der Druck-Ausgabe 15. August 2018 Gesehen am 03.06.2023 |
| Physical Description: | Online Resource |
| ISSN: | 1940-9818 |
| DOI: | 10.2144/98245dt02 |