CHROMOSOME 5 SPECIFIC STSS AND NON-CHIMERIC YAG LIBRARIES

5 号染色体特异性 STSS 和非嵌合 YAG 文库

• 批准号: 3779444
• 负责人: ROBERT MOYZIS
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 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/3779444
• 关键词: artificial chromosomes; chromosomes; cri du chat syndrome; flow cytometry; genetic library; genetic mapping; genetic markers; human genetic material tag; human tissue; in situ hybridization; molecular cloning; nucleic acid sequence; polymerase chain reaction; pulsed field gel electrophoresis; sequence tagged sites; southern blotting

The short term goal of this project is the rapid generation of a "framework" (0.5-1 Mb) sequenced-tagged-site (STS) map of human chromosome 5, "anchored" with non-chimeric yeast artificial chromosome (YAC) clones. To date, 50 chromosome 5 STSs have been developed from M13 clones generated from flow-sorted DNA, and regionally mapped, in collaboration with Dr. John Wasmuth, UC-Irvine. Additional markers will be generated until approximately 300 STSs are mapped. In parallel, the construction of chromosome 5 specific non-chimeric YAC libraries will be pursued, to eliminate drawbacks associated with first generation total genomic YAC libraries, such as the high (>50%) frequency of chimeric DNA. In pilot studies, chromosome specific YAC libraries for human chromosomes 16 and 21 have been constructed. By 1) maximizing the percentage of fragments with two ligatable ends, 2) performing yeast transformations with less than saturating amounts of DNA, and 3) adding an excess of carrier DNA, YAC libraries with less than a few percent chimeric clones were obtained (0/115 analyzed). A framework STS map at the proposed resolution, with accompanying non- chimeric YAC clones, will cover approximately 60% of chromosome 5, and will form the basis for further contig assembly. At Los Alamos, we will concentrate on the short arm of chromosome 5 (50 Mb). Special emphasis will be placed in the region of chromosome 5 involved in the Cri du chat syndrome, one of the most common terminal deletion syndromes in humans. Given our relevant experience with physical mapping of chromosome 16, the construction of a 2 Mb contig map of the short arm of chromosome 5, with 0.1 Mb STS markers, (the 5-year goal of the hUman Genome Project), can be achieved during the requested funding period.

该项目的短期目标是快速生成 人类的“框架”(0.5-1 Mb) 序列标记位点 (STS) 图 5 号染色体，“锚定”非嵌合酵母人工染色体 （YAC）克隆。 迄今为止，已开发出 50 个 5 号染色体 STS M13 克隆由流式分选 DNA 生成，并进行区域定位， 与加州大学欧文分校的 John Wasmuth 博士合作。 额外的标记将 直到映射大约 300 个 STS。 与此同时， 5号染色体特异性非嵌合YAC文库的构建将是 追求，以消除与第一代总数相关的缺点 基因组 YAC 文库，例如嵌合频率高（>50%） 脱氧核糖核酸。 在试点研究中，人类染色体特异性 YAC 文库 16号和21号染色体已经构建完成。 通过 1) 最大化 具有两个可连接末端的片段的百分比，2) 高性能酵母 用少于饱和量的 DNA 进行转化，以及 3) 添加 载体 DNA 过量，YAC 文库含量不到百分之几 获得嵌合克隆（分析0/115）。 拟议决议中的框架 STS 地图，以及随附的非 嵌合 YAC 克隆，将覆盖约 60% 的 5 号染色体，并且 将构成进一步重叠群组装的基础。 在洛斯阿拉莫斯，我们将 集中于 5 号染色体的短臂 (50 Mb)。 特别强调 将被放置在参与 Cri du chat 的 5 号染色体区域 综合征，人类最常见的末端缺失综合征之一。 鉴于我们在 16 号染色体物理绘图方面的相关经验， 构建 5 号染色体短臂的 2 Mb 重叠群图， 具有 0.1 Mb STS 标记（人类基因组计划的 5 年目标）， 可以在请求的资助期内实现。