Ordering of Chromosome Specific Overlapping Cosmid Clones

在拟议的第二阶段工作中，我们将确定来自粘粒文库的10,000个克隆之间的重叠关系，该文库来自部分消化的、染色体分类的人类DNA。这些信息将允许创建有序的染色体特异性文库。这个文库以及后续的其他文库将是人类基因组图谱和测序项目不可或缺的工具。第二阶段的工作将创建一个有序的粘粒文库以及工具包、设备和说明手册，其他研究人员可以使用这些工具在整个人类染色体大小的DNA片段上实现同样的结果。第二阶段的工作包括使用第一阶段工作中开发的“笔线”方法，在所有组合中独立地将大量粘粒克隆的插入片段相互杂交。“笔线”方法允许将所有克隆的DNA分别和独立地与从同一组克隆产生的所有探针进行离散杂交，因此，允许同时确定该组中所有克隆之间的重叠。该方法比目前的可重复协议快几倍。在第二阶段的工作中，我们建议将该方法用于来自20号染色体的重叠克隆文库。

In the phase II work proposed research we will determine the overlapping relationships between-10,000 clones from a cosmid library derived from partially-digested, chromosome-sorted human DNA. This information will allow the creation of an ordered chromosome specific library. This library, and others to follow, will be indispensable tools for the human genome mapping and sequencing project. The phase II work will create an ordered cosmid library as well as kits, devices, and instruction manuals that can be used by other investigators to accomplish the same result on segments of DNA as large as entire human chromosomes. The phase II work involves cross-hybridization of the inserts from a large number of cosmid clones to each other independently in all combinations using the "pen-line" method developed in phase I work. The "pen-line" method allows discrete hybridization of all cloned DNAs separately and independently to all probes generated from the same set clones and, therefore, allows for simultaneous determination of the overlap between all clones in the set. The method is several fold faster than current repeatable protocol. In phase II work, we propose to use the method on an overlapping clone library derived from chromosome 20.