Method of Ordering Chromosome-Specific Overlapping Clones

染色体特异性重叠克隆的排序方法

• 批准号: 9061211
• 负责人: William MacConnell
• 金额: $ 4.97万
• 依托单位: MacConnell Research
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1991-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9061211&HistoricalAwards=false
• 关键词: Method; Ordering; Chromosome; Specific; Overlapping

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.

在拟议的研究中，我们将通过确定源自 DNA 部分消化的克隆之间的重叠关系，开发用于绘制非常大的 DNA 片段的方法和产品。 该方法将创造从与整个人类染色体一样大的DNA片段衍生的大量部分消化的粘粒克隆的端到端排序的可能性。 所提出的方法涉及通过将克隆的DNA或从克隆产生的探针应用于短线片段，将来自大量粘粒克隆的插入片段在所有组合中彼此独立地交叉杂交。 这些线随后按顺序编织成布料。 布中相互交叉的线的交叉点允许所有克隆的 DNA 分别且独立地与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。 所提出的方法如果实施，对于其预期目的将非常强大，并将将克隆订购任务减少为简化、标准化且易于重复的协议。 在第一阶段的工作中，我们建议在大肠杆菌基因组上尝试该方法，以便在尝试从预先存在的流式排序、染色体特异性文库中确定整个人类染色体重叠克隆之前，可以验证和优化该程序。