PHYSICAL MAPPING OF HUMAN CHROMOSOME 12 BY FISH

该项目的总体目标是协助建设 人类12号染色体的详细物理和遗传图谱， 荧光原位杂交技术（FISH） 光学成像显微镜 我们最初的目标是 在中期使用FISH将一组200-300个粘粒克隆至12号染色体 染色体扩散。 这些克隆将提供初始框架图 12号染色体和成核位点，用于进一步的克隆分离。 铝- 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制成的Alu-PCR产物的网格阵列 引物，来自CEPH大插入YAC中的单个YAC克隆 图书馆. 通过该筛选鉴定的单个YAC和YAC重叠群 该过程将在中期染色体上进行FISH定位，以确认 他们的染色体位置，并确定可能存在的 嵌合克隆 重叠群克隆将首先映射为池;当 来自显示嵌合体的重叠群的必要的单个克隆将被 分别映射以识别问题克隆。 多色FISH 然后将使用分析来定义重叠群沿着的相对顺序 染色体和定向成员克隆相对于端粒 和着丝粒。 重叠群之间的缺口大小将通过以下方法测量： 末端克隆与延伸的染色质纤维共杂交， 用拓扑异构酶抑制剂N-[4- （吖啶基氨基）-3-甲氧基苯基]-甲磺酰胺或通过组蛋白 孤立核的耗尽。 后一项研究旨在帮助 在关闭的物理地图，并确定是否粘粒，P-1或 将需要YAC大小克隆以覆盖差距间隔。 最后，阿- 来自相邻YAC重叠群的末端克隆的PCR产物将被扩增。 制备并用于筛选适当的文库，以确定 克隆比跨越这样的间隙间隔。 多色FISH扩展 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.