du Manoir S, Speicher M R, Joos S, Schröck E, Popp S, Döhner H, Kovacs G, Robert-Nicoud M, Lichter P, Cremer T
Institut für Humangenetik, Heidelberg, Germany.
Hum Genet. 1993 Feb;90(6):590-610. doi: 10.1007/BF00202476.
Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with T-cell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed.
比较基因组原位杂交（CGH）为在基因组中搜索失衡的遗传物质提供了新的可能性。从临床或肿瘤标本中制备的标记基因组测试DNA与从具有正常染色体组成的细胞中制备的不同标记的对照DNA混合。混合探针用于与正常中期染色体铺展（CGH中期染色体铺展）进行染色体原位抑制（CISS）杂交。通过不同的荧光染料，如异硫氰酸荧光素（FITC）和异硫氰酸四乙基罗丹明（TRITC），检测杂交后的测试和对照DNA序列。然后，每个染色体或染色体片段的FITC/TRITC荧光强度比值应反映其在测试基因组中与对照基因组相比的相对拷贝数，例如，单体为0.5，二体为1，三体为1.5等。最初，设计模型实验来测试单条染色体上荧光比值测量的准确性。来自多达五个人类染色体特异性质粒文库的DNA用生物素和地高辛配基以不同的半抗原比例进行标记。探针混合物用于与正常人中期染色体铺展进行CISS杂交，并用FITC和TRITC进行检测。配备冷却电荷耦合器件（CCD）相机的落射荧光显微镜用于图像采集。基于商业图像分析软件开发了荧光比值测量程序。对于半抗原比例为4/1、1/1和1/4的情况，单个染色体的荧光比值测量值可作为染色体识别的单一可靠参数。我们的研究结果表明：（1）荧光比值与半抗原比例密切相关；（2）荧光比值测量在多色染色体描绘方面具有潜力。随后，从一名唐氏综合征患者血液、一名T细胞原淋巴细胞白血病患者血液以及一个肾乳头状癌细胞系的培养细胞中制备基因组测试DNA，并应用于CGH实验。正如预期的那样，对于这些测试基因组中以不同拷贝数存在的染色体类型，可以测量到荧光比值的显著差异，包括人类基因组中最小的常染色体21号染色体的三体。此外，不同肿瘤中涉及部分增减的染色体物质可以在CGH中期染色体铺展中映射到其正常的染色体对应物上。基于对CGH中期染色体铺展的CCD图像进行目视检查的另一种更简单的评估程序，也从几位独立观察者那里得到了一致的结果。文中还讨论了CGH分析的陷阱、方法改进及潜在应用。
