Comprehensive microsatellite marker analysis contradicts previous report of segmental maternal heterodisomy of chromosome 14.
Comprehensive microsatellite marker analysis contradicts previous report of segmental maternal heterodisomy of chromosome 14.
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全面的微卫星标记分析与之前关于 14 号染色体节段性母体异二性的报道相矛盾。
DOI：
10.1136/jmg.40.3.e26
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发表时间：
2003
影响因子：
4
通讯作者：
Shaffer,LG
中科院分区：
文献类型：
--
作者：
Coveler,KJ;Sutton,VR;Knox-DuBois,C;Shaffer,LG
方法采用标准方法从患者及父母外周血淋巴细胞中提取基因组DNA。如前所述，我们构建并分析了包含患者母系衍生（“母系”）或父系衍生（“父系”）14号染色体的单染色体体细胞杂交体。6,9我们使用标准PCR扩增D14S261和D14S306标记筛选克隆杂交细胞系，这些标记位于问题区域之外，并鉴定出两个母系（M1和M2）和两个父系（P1和P2）体细胞杂交细胞系（数据未显示）用于进一步分析。这允许母亲和父亲染色体的单倍型的明确构建。然后，我们利用跨越14号染色体长臂的103个高度多态性微卫星标记，对从母亲、孩子、父亲和四个杂交细胞系中提取的基因组DNA进行了全面的微卫星标记分析。如前所述进行直接标记PCR反应，随后进行电泳和放射自显影。10 103个标记中有51个是信息性的（表1）。Martin等人7报道了10个微卫星标记的结果，其中3个显示母体异位或UPD （D14S277、D14S268和D14S77）。
METHODSGenomic DNA was extracted according to standard methods from cultured peripheral blood lymphoblasts from the patient and both parents. We constructed and analysed monochromosomal somatic cell hybrids containing either the patient’s maternally derived (“maternal”) or paternally derived (“paternal”) chromosome 14, as previously described. 6, 9 We screened clonal hybrid cell lines using standard PCR amplification of markers D14S261 and D14S306 that are outside the region in question and identified two maternal (M1 and M2) and two paternal (P1 and P2) somatic cell hybrid cell lines (data not shown) used for further analysis. This allows for unequivocal construction of haplotypes for the maternal and paternal chromosomes. We then performed comprehensive microsatellite marker analysis of genomic DNA extracted from the mother, child, father, and each of the four hybrid cell lines using 103 highly polymorphic microsatellite markers spanning the long arm of chromosome 14. Direct labelled PCR reactions were carried out as previously described, followed by electrophoresis and autoradiography. 10 Fifty-one of the 103 markers were informative (table 1). Martin et al 7 reported results for 10 microsatellite markers, three of which they report as indicating maternal heterodisomy or UPD (14)(D14S277, D14S268, and D14S77).