Direct Isolation of Chromosomal Regions for the Study of Human Genes

工作总结：分离单拷贝基因 从人类基因组中提取是一个费力的过程，因为它依赖于 YAC文库中随机克隆的特征 或者BAC。即使YAC或BAC包含完整的基因， 基因的特异性恢复是一个艰巨的过程。通常一个基因是 可用作一组必须拼接在一起的片段。 我们开发了一种新的直接克隆的新方法 人类染色体片段(TAR克隆)。焦油克隆 方法是基于变换关联重组的 酵母。焦油克隆现已被应用于选择性分离 六个人类基因和基因座(乳腺癌基因、BRCA1和 BRCA2，HPRT基因(有缺陷的人患有Lesch-Nyhan 综合征)，10q25新着丝粒基因座， 染色体Y和D11S12基因座]和一个小鼠的v-Ha-ras 转基因。我们已经证明了用焦油分离的基因 克隆保留了基因的完整性。焦油克隆可用于 循环，提供特定的人类基因和 将它们重新导入人类细胞。一种新的治疗方法 利用TAR选择性克隆基因对 特定染色体的组织与表达研究 区域和基因，包括构建的机会 人类人工染色体与基因治疗。因为只有两个 需要数周时间，TAR克隆提供了新的机会 为了研究人类而直接从个体中研究基因 导致差异的基因突变的多态和鉴定 疾病。在这项工作之前，分离特定的染色体 各地区需要为每个地区开发一个图书馆 研究人员随后进行了广泛的分析，以找到 利息。这些特征表明，焦油克隆可以开辟道路 到全基因的临床研究和基因治疗。

Summary Of Work: Isolation of a single copy gene from a human genome is a laborious process because it relies on the characterization of random clones in libraries comprised of YACs or BACs. Even if a YAC or BAC contains an entire gene, the specific recovery of the gene is an arduous process. Often a gene is available as a contig set of fragments that must be pieced together. We have developed a new novel method for direct cloning of fragment of human chromosomes (TAR cloning). The TAR cloning method is based on Transformation-Associated recombination in yeast. TAR cloning has now been applied for selective isolation of six human genes and loci [breast cancer genes, BRCA1 and BRCA2, the HPRT gene (defective humans have Lesch-Nyhan syndrome), 10q25 neocentromeric locus, the centromeric locus of chromosome Y and the D11S12 locus] and to one mouse v-Ha-ras transgene. We have demonstrated that genes isolated by TAR cloning retain genetic integrity. The TAR cloning can be used in a cycle that provides for specific isolation of human genes and reintroduction of them into human cells. A new procedure for selective cloning of genes by TAR has many utilities for the studying of organization and expression of specific chromosomal regions and genes including the opportunities for construction of human artificial chromosomes and gene therapy. Because only two weeks are required, the TAR cloning provides new opportunities for investigating genes directly from individuals for studying human polymorphism and identification of mutations leading to different diseases. Prior to this work, isolation of specific chromosomal regions would have required the development of a library for each person studied followed by extensive analysis to find the region of interest. These features suggest that TAR cloning can open the way to clinical investigations of entire genes and gene therapy.