DETECTION OF AMPLIFIED DNA-SEQUENCES BY REVERSE CHROMOSOME PAINTING USING GENOMIC TUMOR DNA AS PROBE
DETECTION OF AMPLIFIED DNA-SEQUENCES BY REVERSE CHROMOSOME PAINTING USING GENOMIC TUMOR DNA AS PROBE
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DOI：
10.1007/bf00202475
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发表时间：
1993-02-01
期刊：
影响因子：
5.3
通讯作者：
LICHTER, P
中科院分区：
文献类型：
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作者：
JOOS, S;SCHERTHAN, H;LICHTER, P
用肿瘤细胞的基因组DNA作为复合探针，对正常中期染色体进行原位抑制杂交，对“反向染色体染色”进行了改进。这些探针中所含的扩增DNA序列显示出特异性信号，揭示了这些序列所来源的正常染色体位置。作为一个模型系统，基因组DNA进行了分析，从三个肿瘤细胞系的扩增单位，包括原癌基因c-myc。最小扩增单位约为90 kb，存在16-24个拷贝;最大单位大于600 kb，存在16-32个拷贝。用来自每个细胞系的基因组DNA获得与染色体带8 q24上的不同标记的c-myc探针共定位的特异性信号。在进一步的实验中，在患有多形性胶质母细胞瘤（GBM）的男性患者的情况下使用源自原发性肿瘤材料的基因组DNA。Southern印迹分析使用的表皮生长因子受体基因（EGFR）探针，映射到7 p13表明从这个基因的序列扩增。用反向染色体涂染法，在7 p13和12 q13-q15两条带上都发现了信号。值得注意的是，12 q13-q15上的信号始终更强。较弱的7 p13信号显示与不同标记的EGFR探针的主要信号共定位。在12 q13上观察到该探针的次要信号，表明与EGFR同源的ERB 3序列交叉杂交。结果表明，除了来自条带7 p13的序列外，还存在来自条带12 q13-q15的序列的共扩增。几个癌基因映射到12 q13-q15，为肿瘤相关的原癌基因扩增提供候选基因。尽管扩增序列的性质需要澄清，但本实验证明了用基因组肿瘤DNA进行反向染色体涂染以快速绘制扩增序列所来源的DNA的正常染色体定位的潜力。此外，始终观察到染色体10和X的较弱染色，表明这些染色体仅以一个拷贝存在于GBM基因组中。这种快速方法可用于分析没有来自肿瘤材料的中期扩散的病例。它不需要任何扩增序列的先验知识，可以应用于筛选大量的肿瘤。
A modification of ''reverse chromosome painting'' was carried out using genomic DNA from tumor cells as a complex probe for chromosomal in situ suppression hybridization to normal metaphase chromsome spreads. Amplified DNA sequences contained in such probes showed specific signals, revealing the normal chromosome positions from which these sequences were derived. As a model system, genomic DNAs were analyzed from three tumor cell lines with amplification units including the proto-oncogene c-myc. The smallest amplification unit was about 90 kb and was present in 16-24 copies; the largest unit was bigger than 600 kb and was present in 16-32 copies. Specific signals that co-localized with a differently labeled c-myc probe on chromosome band 8q24 were obtained with genomic DNA from each cell line. In further experiments, genomic DNA derived from primary tumor material was used in the case of a male patient with glioblastoma multiforme (GBM). Southern blot analysis using an epidermal growth factor receptor gene (EGFR) probe that maps to 7p13 indicated the amplification of sequences from this gene. Using reverse chromosome painting, signals were found both on band 7p13 and bands 12q13-q15. Notably, the signal on 12q13-q15 was consistently stronger. The weaker 7p13 signal showed co-localization with the major signal of the differently labeled EGFR probe. A minor signal of this probe was seen on 12q13, suggesting cross-hybridization to ERB3 sequences homologous to EGFR. The results indicate co-amplification of sequences from bands 12q13-q15, in addition to sequences from band 7p13. Several oncogenes map to 12q13-q15 providing candidate genes for a tumor-associated proto-oncogene amplification. Although the nature of the amplified sequences needs to be clarified, this experiment demonstrates the potential of reverse chromosome painting with genomic tumor DNA for rapidly mapping the normal chromosomal localization of the DNA from which the amplified sequences were derived. In addition, a weaker staining of chromosomes 10 and X was consistently observed indicating that these chromosomes were present in only one copy in the GBM genome. This rapid approach can be used to analyze cases where no metaphase spreads from the tumor material are available. It does not require any preknowledge of amplified sequences and can be applied to screen large numbers of tumors.