Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.
Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.
复制标题
630 kb 人类染色体外结构的细菌人工染色体克隆和定位。
作者：
Wang,M;Shouse,S;Lipes,B;Kim,UJ;Shizuya,H;Lai,E
我们利用细菌人工染色体（BAC）克隆系统克隆并绘制了一个630kb的圆形人染色体外结构（称为扩增体）。通过集落杂交从扩增体富集文库中分离出21个bac。插入大小从25到143 kb不等，平均大小为82 kb。扩增体在克隆中的覆盖率约为2.7倍。为了构建扩增体的物理图谱，我们使用了三种不同但互补的方法：杂交、STS内容图谱和指纹图谱。此外，我们比较了这些技术在扩增体bac定位中的优点和缺点。将21个BAC分组为两个contigs，通过筛选人类基因组BAC文库填补了两个小空白（3.5和26.5 kb）。基于bac的物理图谱所揭示的扩增体的组织与之前报道的远程限制图谱一致。我们的研究结果表明，使用BAC系统可以快速克隆630-kb的区域并将其映射到contigs中。由于嵌合和重排的频率较低（<0.1%），这些BAC克隆已准备好进行DNA测序和功能分析。
We have cloned and mapped a circular 630-kb human extrachromosomal structure (termed amplisome) using the bacterial artificial chromosome (BAC) cloning system. Twenty-one BACs were isolated from an amplisome-enriched library by colony hybridization. The insert sizes range from 25 to 143 kb, with an average size of 82 kb. The coverage of the amplisome in clones is approximately 2.7-fold. To construct a physical map of the amplisome, we used three different but complementary methods: hybridization, STS content mapping, and fingerprinting. In addition, we compared the advantages and the drawbacks of these techniques in mapping the amplisomal BACs. The 21 BACs were grouped into two contigs and the two small gaps (3.5 and 26.5 kb) were filled by screening of a human genomic BAC library. The organization of the amplisome revealed by the BAC-based physical map is consistent with the long-range restriction map reported previously. Our results demonstrate that a 630-kb region can be rapidly cloned and mapped into contigs by use of the BAC system. Because of the low frequency (<0.1%) of chimerism and rearrangement, these BAC clones are ready for DNA sequencing and functional analysis.