PHYSICAL MAPPING OF HUMAN CHROMOSOME 12 BY FISH

该项目的总体目标是协助建设 使用人类12号染色体的详细物理和遗传图谱 高分辨率荧光原位杂交(FISH) 光学成像显微镜。我们最初的目标是绘制物理地图 利用FISH技术对12号染色体进行200-300个粘粒克隆 染色体就会传播。这些克隆将提供初始框架图 12号染色体和成核部位，以进行进一步的克隆分离。Alu- 将从这些克隆中的每一个产生PCR产物并用于 筛选用相同Alu生产的Alu-PCR产物的网格阵列 来自CEPH大插入体YAC的单个YAC克隆的引物 图书馆。通过该筛选鉴定出的单个YAC和YAC重叠群 过程将被FISH定位在中期染色体上以确认 它们的染色体位置，并确定可能存在的 嵌合克隆人。重叠克隆将首先映射为池；何时 来自显示嵌合体的重叠群的必要个体克隆将是 单独映射，以识别有问题的克隆。五彩鱼 然后将使用分析来定义重叠群的相对顺序 染色体，并使成员克隆相对于端粒定向 和着丝粒。重叠群之间的间隙大小将通过以下方式测量 末端克隆与延伸的染色质纤维的共杂交 拓扑异构酶抑制剂N-[4-]对细胞的作用 (acridenylamino)-3-methoxyphenyl]-methanesulfonamide或组蛋白 孤立核的耗尽。后一项研究旨在帮助 在物理地图的闭合中，并确定是否一个COSMID、P-1或 需要YAC大小的克隆来覆盖间隔时间。最后，阿卢- 来自相邻YAC重叠群末端克隆的PCR产物将是 准备并用于筛选适当的文库，以便识别 克隆比跨越这样的间隔时间更长。五颜六色的鱼要延伸 染色质纤维将用于确认闭合。

The overall objective of this project is to assist in the construction of a detailed physical and genetic map of human chromosome 12 using fluorescence in situ hybridization (FISH) in consort with high resolution optical imaging microscopy. Our initial goal will be to physically map a set of 200-300 cosmid clones to chromosome 12 using FISH on metaphase chromosome spreads. These clones will provide the initial framework map of chromosome 12 and nucleation sites for further clone isolation. Alu- PCR products will be generated from each of these clones and used to screen a gridded array of Alu-PCR products, made with the same Alu primer, from individual YAC clones in the CEPH large insert YAC libraries. Individual YACs and YAC contigs identified via this screening process will be FISH mapped on metaphase chromosomes to both confirm their chromosomal location and to determine the possible presence of chimeric clones. Contig clones will be first mapped as a pool; when necessary individual clones from contigs exhibiting chimerisms will be mapped separately in order to identify problem clones. Multicolor FISH analysis will then be used to define the relative order of contigs along the chromosome and to orient the member clones relative to the telomere and centromere. The size of gaps between contigs will be measured by cohybridization of end clones to extended chromatin fibers produced by the treatment of cells with the topoisomerase inhibitor N-[4- (acridenylamino)-3-methoxyphenyl]-methanesulfonamide or by histone depletion of isolated nuclei. The latter studies are designed to assist in closure of the physical map and to establish whether a cosmid, P-1 or YAC size clone will be required to cover the gap interval. Finally, Alu- PCR products from the terminal clones of adjacent YAC contigs will be prepared and used to screen an appropriate library in order to identify clones than span such gap intervals. Multicolor FISH to extended chromatin fibers will be used to confirm closure.