Kim U J, Shizuya H, Sainz J, Garnes J, Pulst S M, de Jong P, Simon M I
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Genet Anal. 1995 Oct;12(2):81-4. doi: 10.1016/1050-3862(95)00122-0.
We have previously demonstrated the capability of the Fosmid vector based on Escherichia coli F-factor replicon to stably propagate cosmid-sized human genomic DNA fragments. Using the Fosmid vector, we have constructed and arrayed a 10 x human chromosome 22-specific library, partly by picking human positive clones from a total Fosmid library constructed using DNA from human-hamster hybrid cell line containing human chromosome 22, and partly by using flow-sorted chromosomal DNA. The clones and physical contig maps of the clones in the library will serve as a valuable resource for detailed analysis of the chromosome by providing reliable materials for high resolution mapping and sequencing. In order to efficiently build physical maps for the chromosomal regions of interest spanning several hundred kilobases to a megabase, it is necessary to rapidly identify subsets of the Fosmid clones from the library that cover such regions. In this report, we describe a method of using random amplification products derived from YAC clones to rapidly identify a subset of Fosmid clones that cover a specific genomic subregion.
我们之前已经证明了基于大肠杆菌F因子复制子的Fosmid载体能够稳定地扩增黏粒大小的人类基因组DNA片段。利用该Fosmid载体，我们构建并排列了一个10倍覆盖人类22号染色体的特异性文库，部分是通过从使用含人类22号染色体的人-仓鼠杂交细胞系DNA构建的总Fosmid文库中挑选人类阳性克隆，部分是通过使用流式分选的染色体DNA。文库中的克隆以及这些克隆的物理重叠群图谱将为染色体的详细分析提供有价值的资源，通过提供用于高分辨率定位和测序的可靠材料。为了有效地构建跨越几百千碱基到1兆碱基的感兴趣染色体区域的物理图谱，有必要从文库中快速鉴定出覆盖这些区域的Fosmid克隆子集。在本报告中，我们描述了一种利用源自酵母人工染色体（YAC）克隆的随机扩增产物来快速鉴定覆盖特定基因组子区域的Fosmid克隆子集的方法。
