HIGH-RESOLUTION MULTICOLOR FLUORESCENCE IN-SITU HYBRIDIZATION USING CYANINE AND FLUORESCEIN DYES - RAPID CHROMOSOME IDENTIFICATION BY DIRECTLY FLUORESCENTLY LABELED ALPHOID DNA PROBES

We tested DNA probes directly labeled by fluorescently labeled nucleot ides (Cy3-dCTP, Cy5-dCTP, FluorX-dCTP) for high resolution uni- and mu lticolor detection of human chromosomes and analysis of centromeric DN A organization by in situ hybridization. Alpha-satellite DNA probes sp ecific to chromosomes 1, 2, 3, 4 + 9, 5 + 19, 6, 7, 8, 10, 11, 13 + 21 , 14 + 22, 15, 16, 17, 18, 20, 22, X and Y were suitable for the accur ate identification of human chromosomes in metaphase and interphase ce lls. Cy3-labeled probes had several advantages: (1) a high level of fl uorescence (5-10 times more compared with fluorescein-labeled probes); (2) a low level of fluorescence in solution, allowing the detection o f target chromosomes in situ during hybridization without the washing of slides; and (3) high resistance to photobleaching during prolonged (1-2 h) exposure to strong light, thus allowing the use of a high ener gy mercury lamp or a long integration time during image acquisition in digital imaging microscopy for the determination of weak signals. For di- and multicolor fluorescence in situ hybridization (FISH), we succ essfully used different combinations of directly fluorophorated probes with preservation of images by conventional microscopy or by digital imaging microscopy. FluorX and Cy3 dyes allowed the use of cosmid prob es for mapping in a one-step hybridization experiment. Cyanine-labeled fluorophorated DNA probes offer additional possibilities for rapid ch romosome detection during a simple 15-min FISH procedure, and can be r ecommended for basic research and clinical studies, utilizing FISH.