Labelling quality and chromosome morphology after low temperature FISH analysed by scanning far‐field and near‐field optical microscopy

• DOI: 10.1046/j.1365-2818.2003.01101.x
• Publication Date: 2003-03-12T06:35:06Z
• Authors: R. Winkler; B. Perner; A. Rapp; M. Durm; C. Cremer; K.‐O. Greulich; M. Hausmann
• Keywords: Denaturation (fissile materials)

Summary A non‐enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context ‘low temperature’ means that the denaturation of chromosomal target DNA usually by heat treatment chaotropic agents such as formamide completely omitted so complete reaction took place at 37 °C. For LTFISH, probe had be single‐stranded, which achieved separate thermal only. The pUC1.77 used for all LTFISH experiments. labelling quality (number binding sites, relative background intensity, intensity major minor sites) analysed confocal laser scanning microscopy (CLSM). An optimum specificity signal obtained 15 h time. condition morphology near‐field optical (SNOM). results were compared with chromosomes after (a) centromeres using same chemical FISH but application denaturation, (b) a standard protocol including target. Depending on FISH‐procedure applied, SNOM images show substantial differences chromosome morphology. After appeared much better preserved than FISH. contrast, accompanied very destructive influence indicate that, least certain probes, specific can without target, resulting an apparently well chromatin visualized SNOM. may therefore useful technique whenever regions. Binding mechanisms single‐stranded probes double‐stranded targets are discussed.