HIGH DENSITY GENETIC AND PHYSICAL MAPPING REAGENTS

该提案描述了用于生产高密度的多组聚合物的方法。 用于遗传和物理绘图的试剂，使用我们最近开发的 染色体区域特异性探针的制备和筛选方法 整个细菌人工染色体（BAC）文库， 实验通过显微切割染色体制备探针 来自标准细胞遗传学制备物的感兴趣区域，并使用 简并引物PCR。利用荧光原位筛选BAC 在称为MICROGENETICS TM的程序中进行杂交： 附着在显微镜载玻片上，或者以随机模式，或者以 定义的阵列，并复制;复制品裂解并原位固定， 与荧光探针杂交。对应阳性的细胞 信号是从原始载玻片中"拾取"的。我们将制定战略 用于转移目前存在于多孔阵列中的BAC文库， 将平板置于显微镜载玻片上， 方法，以便可以用一个以上的区域筛选文库 特殊探针同时在区域特异性BAC组被 鉴定后，将其亚克隆并用重复序列筛选 探针;获得重复序列的序列，并获得用于PCR的引物组 扩增被开发用于多态性和遗传分析。的 BAC被组装成重叠群，并且微卫星被物理地 沿着重叠群定位。我们建议开发这些试剂， 生物医学和农业物种的特定区域， 不太可能从大规模的基因组研究中受益。 拟定商业应用： 商业潜力在于AGL与 以及制药和农业行业， 生物医学感兴趣的基因（在大鼠中）或经济性状基因座 (in牛的或猪的）。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将被 主要用于遗传诊断目的的动物育种者（即性状 测试）。区域特定试剂的较小市场在于该领域 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.