SIMULTANEOUS VISUALIZATION OF 7 DIFFERENT DNA PROBES BY INSITU HYBRIDIZATION USING COMBINATORIAL FLUORESCENCE AND DIGITAL IMAGING MICROSCOPY
SIMULTANEOUS VISUALIZATION OF 7 DIFFERENT DNA PROBES BY INSITU HYBRIDIZATION USING COMBINATORIAL FLUORESCENCE AND DIGITAL IMAGING MICROSCOPY
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DOI：
10.1073/pnas.89.4.1388
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发表时间：
1992-02-15
影响因子：
11.1
通讯作者：
WARD, DC
中科院分区：
文献类型：
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作者：
RIED, T;BALDINI, A;WARD, DC
探针的组合标记（即，具有两种或多种不同的报道分子）增加了可通过荧光原位杂交同时检测的靶序列的数目。我们已经使用了一个epifluorescence显微镜配备了一个数字成像相机和计算机软件的假彩色和合并图像，以区分多达七个不同的探针，仅使用三种荧光染料。染色体特异性的着丝粒重复克隆和染色体特异性的“复合”探针集通过PCR产生，其中掺入了不同的修饰核苷酸混合物，包括荧光素缀合的dUTP。通过切口平移类似地标记粘粒克隆。该技术已被用于描绘7个不同的人类染色体的着丝粒，在4 '，6-diamidino-2-phenylindole染色的中期铺展和间期核，映射6粘粒克隆在一个单一的杂交实验和检测染色体易位染色体涂。多参数杂交分析有助于分子细胞遗传学、基于探针的病原体诊断和基因定位研究。
Combinatorial labeling of probes (i.e., with two or more different reporters) increases the number of target sequences that can be detected simultaneously by fluorescence in situ hybridization. We have used an epifluorescence microscope equipped with a digital imaging camera and computer software for pseudocoloring and merging images to distinguish up to seven different probes using only three fluorochromes. Chromosome-specific centromere repeat clones and chromosome-specific "composite" probe sets were generated by PCR in which different mixtures of modified nucleotides, including fluorescein-conjugated dUTP, were incorporated. Cosmid clones were labeled similarly by nick-translation. The technique has been used to delineate the centromeres of seven different human chromosomes, on both 4',6-diamidino-2-phenylindole-stained metaphase spreads and interphase nuclei, to map six cosmid clones in a single hybridization experiment and to detect chromosome translocations by chromosome painting. Multiparameter hybridization analysis should facilitate molecular cytogenetics, probe-based pathogen diagnosis, and gene mapping studies.