Mrasek Kristin, Wilhelm Kathleen, Quintana Luciana G, Theuss Luise, Liehr Thomas, Leskovac Andreja, Filipovic Jelena, Joksic Gordana, Joksic Ivana, Weise Anja
Jena University Hospital, Institute of Human Genetics, Friedrich Schiller University, Kollegiengasse 10, Jena, D-07743, Germany.
Methods Mol Biol. 2015;1227:289-98. doi: 10.1007/978-1-4939-1652-8_15.
Genomic instability tends to occur at specific genomic regions known as common fragile sites (FS). FS are evolutionarily conserved and generally involve late replicating regions with AT-rich sequences. The possible correlation between some FS and cancer-related breakpoints emphasizes on the importance of understanding the mechanisms of chromosomal instability at these sites. Although about 230 FS have already been mapped cytogenetically, only a few of them have been characterized on a molecular level. In this chapter, we provide a protocol for mapping of common FS using bacterial artificial chromosome (BAC) probes in fluorescence in situ hybridization (FISH) and suggest the usage of lymphocytes from Fanconi anemia patients as a model system. In the latter, rare FS are expressed much more frequently than in, for example, aphidicolin-induced blood lymphocyte preparations. Knowing the exact location of FS enables the molecular comparison of their location and breakpoints that appear during evolution, cancer development and inherited disorders.
基因组不稳定倾向于发生在特定的基因组区域，即所谓的常见脆性位点（FS）。FS在进化上是保守的，通常涉及富含AT序列的晚复制区域。一些FS与癌症相关断点之间的可能关联强调了理解这些位点染色体不稳定机制的重要性。尽管已经通过细胞遗传学方法定位了约230个FS，但其中只有少数在分子水平上得到了表征。在本章中，我们提供了一种使用细菌人工染色体（BAC）探针进行荧光原位杂交（FISH）来定位常见FS的方案，并建议使用范可尼贫血患者的淋巴细胞作为模型系统。在后者中，罕见的FS比例如阿非迪霉素诱导的血液淋巴细胞制剂中更频繁地表达。了解FS的确切位置能够对其在进化、癌症发展和遗传性疾病过程中出现的位置和断点进行分子比较。
