HUMAN GENOME CLONING AND ISOLATION OF SPECIFIC DNAS IN YEAST

人类基因组克隆和酵母中特定 DNA 的分离

• 批准号: 6106745
• 负责人: MICHAEL A RESNICK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6106745
• 关键词: DNA; DNA repair; DNA replication; Escherichia coli; Saccharomyces cerevisiae; bacterial genetics; fungal genetics; genetic recombination; genome; human; human genetic material tag; molecular cloning; nucleic acid repetitive sequence

Summary of Work: The Human Genome Project has made great strides in the decade since its inception including the cloning of most of the chromosomal DNA, the identification of a unique sequence (STS) approximately every 100 kb and the sequencing of short regions of nearly all expressed genes (EST's). Critical in the characterization of the human genome has been the cloning of large chromosomal fragments, which has traditionally involved the isolation of random DNA fragments into vectors followed by bacteria or yeast. Previously we described a novel nonenzymatic method for cloning large fragments of human DNA into yeast based on Transformation- Associated Recombination (TAR cloning) that we applied to the direct isolation of specific DNAs. TAR cloning has now been applied to the selective isolation as linear and/or circular yeast artificial chromosomes (YACs) of i) human DNA from a radiation hybrid rodent cell line, ii) specific isolation of genes and regions directly from total human DNA including rDNA genes, and single copy genes BRCA1, BRCA2, and HPRT. It is being applied to the isolation from mouse cells of an element that is responsive to environmental agents. We have established that TAR cloned genes can be fully functional based on the isolation of HPRT and transfer back to an hprt- cell line. Thus, TAR cloning and opportunities to modify the isolated material enables genetic modification and correction. In addition we have established that a gene region can be isolated with only sequence information from one end of the gene, as one TAR "hook" and a common repeat as the other hook, thereby increasing the opportunities to examine regions around a gene. Current directions also include assessment of minimum size of sequence needed for TAR cloning and refinement of clone detection. Since only a few weeks are required, TAR cloning provides many opportunities for investigating genes and chromosomal regions directly from individuals. It can be used for studying human polymorphisms, clinical diagnosis, gene therapy and the filling-in of gaps in sequenced regions.

工作摘要：人类基因组计划 自成立以来的十年间取得了巨大进步，包括 克隆大部分染色体DNA，鉴定 大约每 100 kb 就有一个独特序列 (STS)，并且 对几乎所有表达基因 (EST) 的短区域进行测序。 人类基因组表征的关键是 传统上，大染色体片段的克隆 涉及将随机 DNA 片段分离到载体中 其次是细菌或酵母。之前我们介绍过一部小说 克隆人类 DNA 大片段的非酶法 基于转化相关重组转化为酵母 （TAR 克隆）我们应用于特定的直接分离 DNA。 TAR克隆现已应用于选择性分离 作为 i) 的线性和/或环状酵母人工染色体 (YAC) 来自放射杂交啮齿动物细胞系的人类 DNA，ii) 特异性 直接从人类总 DNA 中分离基因和区域 包括rDNA基因和单拷贝基因BRCA1、BRCA2、 和HPRT。它被应用于从小鼠细胞中分离 对环境因素做出反应的元素。我们有 确定 TAR 克隆基因可以完全发挥功能 HPRT 的分离并转移回hprt-细胞系。因此， TAR 克隆和修改分离材料的机会 实现基因改造和修正。此外我们还有 确定仅用序列即可分离基因区域 来自基因一端的信息，作为一个 TAR“钩子”和一个 与另一个钩子共同重复，从而增加 检查基因周围区域的机会。当前方向 还包括评估所需的最小序列大小 TAR 克隆和克隆检测的改进。由于只有少数 TAR 克隆提供了许多机会 直接研究基因和染色体区域 个人。可用于研究人类多态性， 临床诊断、基因治疗和填补空白 测序区域。