应用FISH技术鉴定肿瘤CGH微阵列BAC克隆

Identification of BAC clones for array CGH in human cancer by FISH technique

目的:鉴定用于肿瘤比较基因组杂交(CGH)微阵列细菌人工染色体(BAC)克隆的质量。方法:常规法制备健康自愿者外周血染色体标本;BAC(获自RPCI-11文库和CTC文库)定位信息参考UCSC和NCBI资源数据库编辑;探针用切口平移法分别标记上生物素-16-dUTP或地高辛-11-dUTP;各BAC克隆的精确定位在正常人中期分裂相,应用荧光原位杂交(FISH)技术进行确认。选择在肿瘤发生、发展过程中有意义的223种BAC克隆,分析它们在染色体相应区域的拷贝数和分子构成。结果:FISH分析表明:正常BAC克隆占81.62%(186/223),出现额外FISH杂交信号的BAC克隆占13.45%(30/223),定位信息错误的BAC克隆占3.58%(8/223),BAC克隆大肠菌扩增失败占1.35%(3/223)。结论:BAC克隆作为探针点样制备CGH微阵列时,FISH法鉴定是必要的。

Objective To evaluate the quality of BAC clones for CGH microarrays in the detection of tumors. Methods Chromosome preparations were made from peripheral blood of the healthy volunteers in the conventional manner. The locations of BAC （RPCI-11 library and CTC library） within the region of interest were compiled from information archived by the UCSC and the NCBI. Probes were labeled by nick-translation with biotin-16-dUTP or digoxigenin-11-dUTP. Precise localization of each BAC was confirmed using normal metaphase chromosomes by FISH technique. The copy number and molecular organization of the region of 223 BAC clones which were crucial in the development and progression of human cancers were investigated. Results The FISH analysis indicated the normal BAC clones accounted for 81.62% of the total （186/223） % the abnormal clones with additional FISH noises accounted for 13.45% （30/223）; those with wrong localization pattern was 3.58% （8/223）, and those with no bacterial growth was 1.35% （3/223）, respectively. Conclusion FISH technique is effective and useful in the identification of BAC clones for array CGH.