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一个新的微卫星多态标记 D14S1435 的FISH定位 |
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郑其平 余龙 张民 胡培蓉 毛宁辉 许月芳 赵寿元
【摘要】 目的 将从人染色体14q24.3显微切割制备的DNA文库中分离得到的一个新的微卫星多态标记(D14S1435，CA重复序列，人群中存在11种等位形式，PIC值为0.85)进行染色体荧光原位杂交(FISH)返回定位。 方法 以此多态标记筛选人Lambda/DASH基因组文库，得到的阳性重组噬菌体DNA，通过BamH Ⅰ完全酶切，低熔点胶电泳回收插入片段，再经Sau3A Ⅰ酶切，接头捕获PCR(linker-catch PCR)法标记探针进行FISH。 结果 将这一新的STR精确地定位于人染色体14q24.3，证实了从染色体显微切割探针池分离染色体区带特异性遗传标记的可靠性。 结论 经染色体显微切割、PCR及微克隆方法所得到的遗传标记，通过染色体荧光原位杂交证实其确实来自所切割区域，增加了这一区域可用于连锁分析的遗传标记，为定位在14q24.3区带内若干遗传病的基因诊断和基因克隆，提供一个新的有价值的遗传标记。
【关键词】 人染色体14q24.3 微卫星多态(CA重复序列) 荧光原位杂交
CHROMOSOME ASSIGNMENT OF A NOVEL MICROSATELLITE POLYMORPHIC MARKER (D14S1435) BY FLUORESCENCE IN SITU HYBRIDIZATION Zheng Qiping, Yu Long*, Zhang Min, Hu Peirong, Mao Ninghui, Xu Yuefang, Zhao Shouyuan.* State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433 P.R.China
【Abstract】 Objective Chromosome assignment of a novel microsatellite marker (D14S1435 containing CA repeats and showing 11 alleles and 0.85 of PIC value in Chinese population) isolated from microdissected human chromosome 14q24.3 DNA library by fluorescence in situ hybridization(FISH). Methods The polymorphic marker was used as probe to screen a human Lambda/DASH genome library, a positive clone was isolated and the recombinant phage DNA was extracted as primary material. The insert was recovered through BamH Ⅰ digestion to complete and low melting point agarose gel electrophoresis, after processed with Sau3A Ⅰ the probe was prepared by linker-catch PCR method and the following FISH was conducted to metaphase chromosomes. Results This novel STR was precisely mapped to human chromosome 14q24.3, and this demonstrated the validity of isolating chromosome band-specific genetic markers from probe pool made by chromosome microdissection. Conclusion Confirmed by chromosome fluorescence in situ hybridization, the novel STR obtained by chromosome microdissection, PCR and subcloning method was really from the microdissected region. Th[1] [2] 下一页
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