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

• Verfasst von: Durm, Markus [VerfasserIn]
• Verfasst von: Schüssler, L. [VerfasserIn]
• Verfasst von: Münch, H. [VerfasserIn]
• Verfasst von: Craig, J. [VerfasserIn]
• Verfasst von: Ludwig, Horst [VerfasserIn]
• Verfasst von: Hausmann, Michael [VerfasserIn]
• Verfasst von: Cremer, Christoph [VerfasserIn]
• Titel: Fast-painting of human metaphase spreads using a chromosome-specific, repeat-depleted DNA library probe
• Verf.angabe: M. Durm, L. Schüssler, H. Münch, J. Craig, H. Ludwig, M. Hausmann, C. Cremer
• Jahr: 1998
• Umfang: 6 S.
• Fussnoten: Elektronische Reproduktion der Druck-Ausgabe 15. August 2018 ; Gesehen am 03.06.2023
• Titel Quelle: Enthalten in: BioTechniques
• Ort Quelle: London, UK : Future Science Ltd, 1983
• Jahr Quelle: 1998
• Band/Heft Quelle: 24(1998), 5, Seite 820-825
• ISSN Quelle: 1940-9818
• DOI: doi:10.2144/98245dt02
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1851470581

Abstract

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.