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: 3757539
• 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/3757539
• 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 人的“框架”（0.5-1 Mb）序列标记位点（STS）图谱 5号染色体，与非嵌合酵母人工染色体“锚定” (YAC)克隆 到目前为止，已经开发了50个5号染色体STS， 从流式分选的DNA产生的M13克隆， 与加州大学欧文分校的约翰·瓦斯科博士合作。 其他标记将 直到大约300个STS被映射。 并行地将第 5号染色体特异性非嵌合YAC文库的构建将 追求，以消除与第一代总 基因组YAC文库，例如高频率（>50%）的嵌合YAC文库， DNA. 在初步研究中，人类染色体特异性YAC文库 16号和21号染色体已经构建。 （1）最大化 具有两个可连接末端的片段的百分比，2）进行酵母 用小于饱和量的DNA转化，和3）加入 过量的载体DNA，YAC文库中含有不到百分之几的 获得嵌合克隆（0/115分析）。 一个框架STS地图在拟议的决议，与伴随的非- 嵌合YAC克隆将覆盖约60%的5号染色体， 将形成进一步叠连群组装的基础。 在洛斯阿拉莫斯，我们将 集中在5号染色体的短臂（50 Mb）上。 特别强调 将被放置在与Cri du chat有关的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号染色体物理定位方面的相关经验， 5号染色体短臂的2 Mb重叠群图谱的构建， 0.1 Mb STS标记（人类基因组计划的5年目标）， 可以在申请资助期间完成。