Wang M, Shouse S, Lipes B, Kim U J, Shizuya H, Lai E
Department of Pharmacology, University of North Carolina at Chapel Hill 27599-7365, USA.
Genome Res. 1996 Jul;6(7):612-9. doi: 10.1101/gr.6.7.612.
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.
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的环状人类染色体外结构（称为扩增体）图谱。通过菌落杂交从一个富含扩增体的文库中分离出21个BAC。插入片段大小从25kb到143kb不等，平均大小为82kb。克隆中扩增体的覆盖度约为2.7倍。为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含量作图和指纹图谱分析。此外，我们比较了这些技术在绘制扩增体BAC图谱方面的优缺点。这21个BAC被分为两个重叠群，通过筛选人类基因组BAC文库填补了两个小间隙（3.5kb和26.5kb）。基于BAC的物理图谱所揭示的扩增体结构与先前报道的长程限制性图谱一致。我们的结果表明，利用BAC系统可以快速将一个630kb的区域克隆并绘制到重叠群中。由于嵌合和重排的频率较低（<0.1%），这些BAC克隆可用于DNA测序和功能分析。
