Nederlof P M, Robinson D, Abuknesha R, Wiegant J, Hopman A H, Tanke H J, Raap A K
Department of Cytochemistry and Cytometry, University of Leiden, The Netherlands.
Cytometry. 1989 Jan;10(1):20-7. doi: 10.1002/cyto.990100105.
A method is described for visualizing three nucleic acid sequences simultaneously by in situ hybridization using a new blue immunofluorescent label, amino methyl coumarin acetic acid (AMCA), in combination with green and red fluorescing FITC and TRITC. Three chromosome-specific repetitive probes labeled with either amino acetyl fluorene (AAF), mercury, or biotin were hybridized simultaneously to metaphase chromosomes prepared from human blood lymphocytes or to interphase tumor nuclei. Conditions for the combined use of three immunocytochemical affinity systems as well as the optimal spectral separation of the three fluorescing labels have been determined. Three-color in situ hybridization was applied to the study of numerical chromosome abnormalities as occur in human solid tumors. Further applications of this method in prenatal diagnosis for the detection of aneuploidy of the most frequently involved autosomes, as well as for the quantification of gene copy number and mRNA expression, are discussed.
本文描述了一种通过原位杂交同时可视化三个核酸序列的方法，该方法使用一种新的蓝色免疫荧光标记物氨基甲基香豆素乙酸（AMCA），并结合绿色荧光的异硫氰酸荧光素（FITC）和红色荧光的四甲基异硫氰酸罗丹明（TRITC）。三种分别用氨基乙酰芴（AAF）、汞或生物素标记的染色体特异性重复探针同时与从人血淋巴细胞制备的中期染色体或间期肿瘤细胞核进行杂交。已经确定了三种免疫细胞化学亲和系统联合使用的条件以及三种荧光标记物的最佳光谱分离。三色原位杂交应用于研究人类实体瘤中出现的染色体数目异常。还讨论了该方法在产前诊断中的进一步应用，用于检测最常涉及的常染色体的非整倍性，以及基因拷贝数的定量和mRNA表达。
