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In all DNA-DNA in situ hybridization (ISH) procedures described so far in the literature, the production of single-stranded target DNA sequences plays a decisive role. This can be achieved either by enzymatic treatment at physiological temperatures or by the separation of double-stranded DNA sequences. Denaturation by heat and chemical agents (e.g. formamide) is regarded as a prerequisite for the non-enzymatic ISH process. However, additional mechanisms of a non-enzymatic ISH procedure are conceivable which do not require high temperature treatment combined with formamide. Here, we report on a non-enzymatic, non-formamide, low temperature, fluorescence in situ hybridization (FISH) procedure which allowed a microscopic visualization and quantitative fluorescence analysis of the binding sites of a repetitive DNA probe. Following only probe denaturation at 94 °C, hybridization was performed at 52 °C for 30 min, i.e., at nearly physiological temperatures. Moreover, increasing the hybridization time to 3 hours indicated that hybridization sites became also visible at 37 °C. Since the protocols are based on recently described Fast FISH developments, the technique will be called Low Temperature Fast-FISH (LTFF).

Non-Enzymatic, Low Temperature Fluorescence in situ Hybridization of Human Chromosomes with a Repetitive α-Satellite Probe