Method of Ordering Chromosome-Specific Overlapping Clones

在拟议的研究中，我们将开发方法和产品，通过确定来自DNA部分消化的克隆之间的重叠关系，来绘制非常大的DNA片段。该方法将创造端到端排序大量部分消化粘粒克隆的可能性，这些克隆来自整个人类染色体大小的DNA片段。所提出的方法包括将大量粘粒克隆的插入片段在所有组合中相互独立地交叉杂交，方法是将克隆的DNA或克隆产生的探针应用于短线段。这些线随后按顺序编织成布。布料中相互交叉的线的交点允许将所有克隆的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，从而允许同时确定该组中的所有克隆之间的重叠。所提出的方法，如果实施，将是非常强大的预期目的，并将减少克隆排序任务为一个简化的，标准化的，易于重复的协议。在第一阶段的工作中，我们建议在大肠杆菌基因组上尝试这种方法，以便在尝试从先前存在的流动排序的染色体特异性文库中确定整个人类染色体重叠克隆之前，可以验证和优化该过程。

In the proposed research we will develop methodologies and products for mapping very large pieces of DNA by determining the overlapping relationships between clones derived from a partial digest of the DNA. The method will create the possibility of ordering end-to-end a large number of partial- digest cosmid clones derived from segments of DNA as large as an entire human chromosome. The proposed method involves crosshybridization of the inserts from a large number of cosmid clones to each other independently in all combinations by applying cloned DNA or probes generated from the clones to short pieces of thread. These threads are subsequently woven in order into cloth. The intersection points of the threads crossing each other in the cloth allows for discrete hybridization of all cloned DNAs separately and independently to all probes generated from the same set of clones and, therefore, allows for simultaneous determination of the overlap between all clones in the set. The proposed method, if implemented, would be very powerful for its intended purpose and would reduce the clone ordering task into a simplified, standardized, and easily repeatable protocol. In phase I work, we propose to try the method on the E.coli genome so that the procedure can be verified and optimized before attempting an entire human chromosome overlapping clone determination from a preexisting flow-sorted, chromosome-specific library.