Yb. Yurov et al., HIGH-RESOLUTION MULTICOLOR FLUORESCENCE IN-SITU HYBRIDIZATION USING CYANINE AND FLUORESCEIN DYES - RAPID CHROMOSOME IDENTIFICATION BY DIRECTLY FLUORESCENTLY LABELED ALPHOID DNA PROBES, Human genetics, 97(3), 1996, pp. 390-398
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.