Multiple fluorescence and reflectance simultaneous detection by confocal microscopy of HaeIII digested DNA sequences

Human metaphase chromosomes were isolated and digested in situ with Haelll restriction enzyme to detect cytosine and guanine-rich sequences (CpG islands), which are known to be associated with most of the mammalian genes. Digested DNA was reconstructed by in situ nick translation employing digoxigenin-labeled nucleotides. The DNA sequences were revealed bg antibodies conjugated either with fluorescein isothiocyanate or l-nm colloidal gold. DNA was counterstained with propidium iodide. A sensitive, high resolution method for visualizing three signals, simultaneously excited by a single argon laser line of 188 nm has been developed. The green fluorescence of fluorescein isothiocyanate was detected in combination,vith the red fluorescence of propidium iodide and the third signal was imaged by employing the reflectance mode of the confocal microscope after silver enhancement of the gold beads. The high reflectance intensity, the accurate localization and the non fading properties of colloidal gold made the reaction a valuable tool for the detection of antigens and, as a consequence. Of specific DNA sequences in chromosome preparations. Overlaying of three signals allowed the simultaneous observation of distinct structures: total DNA, as well as fluorescein- and gold-labeled sequences alter in situ nick translation, or total DNA and centromeric sequences of two different chromosome pairs (17 and X) after in situ hybridization. The use of Haelll restriction enzyme me that rut CpG islands combined with in situ nick translation identified the chromosome sites where active, inactive or housekeeping genes can be located, In chromosomes, the fluorescent reaction pattern showed large areas fabling, while a more defined staining, often organized in spot pairs that resembled an R-like banding, was detected when the reflected mode was used. These results are confirmed by the observation that R-like hands actually are multiple symmetrical spots localized on sister chromatids. In addition some chromosomes, and in particular 1 and 9, displayed a C-negative banding due: to the negativity of the centromeric areas. Reflectance confocal scanning microscopy and in situ nick translation represent a powerful tool to study the in situ genome organization.