DEVELOPMENT OF SYSTEMS FOR RAPID CHROMOSOME MAPPING

我们建议开发简单而通用的系统来绘制哺乳动物的图谱 基因对单个染色体和亚染色体条带的应用 聚合酶链式反应(PCR)技术。放大是 利用通用的聚合酶链式反应模板接头和引物进行 (盒式磁带)被随机插入到染色体DNA中 连接到限制性内切酶消化的组合。一位常任理事国 然后，将建立扩增的染色体DNA的收集 克隆到一个质粒载体上。有了这个系统，染色体图谱就可以 然后以几种方式进行；(1)通过cDNA或基因杂交 用片段探针扩增固定化的染色体DNA 杂交膜，(2)通过对收集的 用基因特异性引物克隆染色体DNA；(3)利用 将所克隆的DNA作为染色体特异性杂交探针。在第一阶段 我们将使用聚合酶链式反应扩增来评估这种方法的可行性。 完整的宝石DNA。接下来是对整体的放大 中期细胞染色体分离以优化操作和 染色体的加工。第二阶段将把方法扩展到 解剖单个染色体并从所有23条染色体中构建文库 人类染色体，以及亚染色体区域的带 特别感兴趣。

We propose to develop simple and versatile systems to map mammalian genes to individual chromosome and subchromosome bands by application of the polymerase chain reaction (PCR) technique. Amplification is performed by utilizing a universal PCR template-adaptor and primer (cassette) which is inserted randomly into the chromosomal DNA by ligation to combinations of restriction enzyme digests. A permanent collection of the amplified chromosomal DNA will then be established by cloning into a plasmid vector. With this system chromosome mapping can then be performed in several ways; (1) By hybridization of cDNA or gene fragment probes to amplified chromosomal DNA immobilized on hybridization membranes, (2) By PCR analysis to collections of the cloned chromosomal DNA with gene specific primer sets, and (3) By use of the cloned DNA as chromosome specific hybridization probes. In phase I we will evaluate the feasibility of this approach using PCR amplified whole gemonic DNA. This will be followed by amplification of whole metaphase cell chromosome dissections to optimize the manipulation and processing of chromosomes. Phase II will extend the methodology to dissected individual chromosomes and construct libraries from all 23 human chromosomes, as well as subchromosomal bands in regions of particular interest.