Szuhai Karoly, Tanke Hans J
Department of Molecular Cell Biology, Leiden University Medical Center, PO Box 9600 (zone S1-P), 2300 RC Leiden, The Netherlands.
Nat Protoc. 2006;1(1):264-75. doi: 10.1038/nprot.2006.41.
Combined binary ratio labeling (COBRA) is designed to increase the multiplicity of fluorescence in situ hybridization (FISH)--i.e., the number of targets that can be distinguished simultaneously. In principle, chemical (ULS), enzymatic (nick translation or random priming) or PCR-based labeling procedures of probes can be used. The method was originally designed to label chromosome-painting probes, but has also been used for probe sets specific for subtelomeric regions. COBRA imaging requires a digital fluorescence microscope equipped for sequential excitation and recording of color images. Staining of all 24 human chromosomes is accomplished with only four fluorochromes, compared with five for methods based on combinatorial labeling. The COBRA procedure takes approximately 6 h laboratory work, 2-3 d incubation and a few hours imaging. The method is routinely applied in research (cultured cells from human or mouse origin) or to support clinical diagnosis, such as postnatal and perinatal genetic testing and in solid tumors.
组合二元比率标记法（COBRA）旨在增加荧光原位杂交（FISH）的多重性，即同时可区分的靶标的数量。原则上，可以使用探针的化学（通用链接系统）、酶促（缺口平移或随机引物法）或基于聚合酶链反应的标记程序。该方法最初设计用于标记染色体涂染探针，但也已用于亚端粒区域特异性的探针组。COBRA成像需要配备用于顺序激发和彩色图像记录的数字荧光显微镜。与基于组合标记的方法使用五种荧光染料相比，仅用四种荧光染料就能完成对人类所有24条染色体的染色。COBRA程序大约需要6小时的实验室工作、2 - 3天的孵育时间以及数小时的成像时间。该方法常规应用于研究（源自人或小鼠的培养细胞）或支持临床诊断，如产后和围产期基因检测以及实体瘤检测。
