HIGH DENSITY GENETIC AND PHYSICAL MAPPING REAGENTS

本提案描述了用于生产高密度集的方法 用于基因和物理作图的试剂，使用我们最近开发的 染色体区域特异性探针的制备方法及筛选 完整的细菌人工染色体(BAC)文库 做实验。这些探针是通过显微切割染色体来制备的 来自标准细胞遗传学准备的感兴趣区域，并用 简并引物聚合酶链式反应。用原位荧光法对细菌进行筛选 被称为微基因TM的杂交过程中的细胞 以随机图案或以 定义的阵列，并复制；复制副本被裂解并就地固定，以及 与荧光探针杂交。与阳性对应的单元格 信号是从原始幻灯片中“挑选”出来的。我们将制定战略 用于传输多井阵列中当前存在的BAC库 在显微镜载玻片上的板和发展组合杂交 方法，以便可以在多个区域筛选文库 同时进行特定的探头。在特定于区域的BAC集被 经鉴定，它们被亚克隆，并用重复序列筛选 获得重复序列，并设置用于聚合酶链式反应的引物 扩增是为多态和遗传分析而开发的。这个 BACs被组装成重叠群，而微卫星在物理上 沿着重叠群定位的。我们建议开发这些试剂用于 具有生物医学和农业价值的物种的特定区域 不太可能从大规模的基因组努力中受益。 建议的商业应用： AGL的商业潜力在于组建合资企业 以及制药和农业行业，以追求特定的 生物医学感兴趣的基因(在大鼠中)或经济性状基因 (牛或猪)。生物医学感兴趣的基因将用于 诊断和/或治疗目的，以及经济性状基因座 主要用于动物繁育者的遗传诊断目的(即性状 测试)。该领域存在一个较小的地区特定试剂市场 所有这些动物的基因组研究。

This proposal describes methods for producing high density sets of reagents for genetic and physical mapping, using our recently developed methods for preparing chromosome region specific probes and screening entire bacterial artificial chromosome (BAC) libraries in one simple experiment. The probes are prepared by microdissecting the chromosome region of interest from standard cytogenetic preps, and amplification with degenerate primer PCR. The BACs are screened using fluorescence in situ hybridization in a procedure referred to as MICROGENETICS TM: the cells are attached to a microscope slide, either in a random pattern or in a defined array, and replicated; the replica is lysed and fixed in situ, and hybridized with the fluorescent probe. Cells corresponding to positive signals are "picked" from the original slide. We will develop strategies for transferring BAC libraries that currently exist in arrays of multiwell plates onto microscope slides and develop combinatorial hybridization methods so that libraries can be screened with more than one region specific probe simultaneously. After region specific sets of BACs are identified, they are subcloned and screened with repetitive sequence probes; sequences of the repeats are obtained, and primer sets for PCR amplification are developed for polymorphism and genetic analyses. The BACs are assembled into contigs, and the microsatellites are physically positioned along the contig. We propose to develop these reagents for specific regions of species of biomedical and agricultural interest that are unlikely to benefit from a massive genome effort. PROPOSED COMMERCIAL APPLICATION: The commercial potential lies in formation of joint ventures between AGL and the pharmaceutical and agricultural industries, in pursuit of specific genes of either biomedical interest (in the rat) or economic trait loci (in bovine or porcine). Genes of biomedical interest will be for diagnostics and/or therapeutic purposes, and economic trait loci will be for animal breeders primarily for genetic diagnostic purposes (i.e. trait testing). A smaller market for region specific reagents lies in the field of genome research on all these animals.