Ashworth L K, Alegria-Hartman M, Burgin M, Devlin L, Carrano A V, Batzer M A
Human Genome Center, Lawrence Livermore National Laboratory, Livermore, California 94551, USA.
Anal Biochem. 1995 Jan 20;224(2):564-71. doi: 10.1006/abio.1995.1088.
The generation of contiguous physical maps is often complicated by a variety of factors including the type of cloning system used. Here we describe procedures for the isolation, rapid characterization, and physical mapping of large-insert recombinant bacterial clones from total human genomic BAC (bacterial artificial chromosome) and PAC (P1-derived artificial chromosome) libraries containing clones with an average insert size of 150 kbp. After initial isolation, the clones were subjected to a variety of fingerprinting procedures including inter-Alu PCR, semiautomated fluorescent finger-printing, and EcoRI restriction fragment mapping. Individual BAC and PAC clones were also used as probes to interrogate arrayed chromosome 19-specific cosmid libraries. The combination of analyses facilitated the identification of chromosome-specific large-insert clones as well as the construction of a large (1.2 Mb) high-resolution BAC, PAC, and cosmid contig in 19q13.2, spanning the region from the carcinoembryonic antigen gene family to the X-ray repair cross complementing 1 DNA repair gene. This type of approach directly demonstrates the utility of large-insert recombinant bacterial clones for the construction of contiguous physical maps of entire chromosomes.
连续物理图谱的构建常常因包括所用克隆系统类型在内的多种因素而变得复杂。在此，我们描述了从平均插入片段大小为150 kbp的人类基因组BAC（细菌人工染色体）和PAC（P1衍生人工染色体）文库中分离、快速鉴定和物理定位大插入片段重组细菌克隆的方法。初步分离后，对这些克隆进行了多种指纹分析程序，包括Alu间PCR、半自动荧光指纹分析和EcoRI限制性片段图谱分析。单个BAC和PAC克隆还被用作探针来检测排列好的19号染色体特异性黏粒文库。这些分析方法的结合有助于鉴定染色体特异性大插入片段克隆，并在19q13.2构建一个大的（1.2 Mb）高分辨率BAC、PAC和黏粒重叠群，该重叠群跨越从癌胚抗原基因家族到X射线修复交叉互补1 DNA修复基因的区域。这种方法直接证明了大插入片段重组细菌克隆在构建整条染色体连续物理图谱方面的实用性。
