YEAST ARTIFICIAL CHROMOSOME-BASED GENOME MAPPING

基于酵母人工染色体的基因组作图

• 批准号: 3106311
• 负责人: DAVID SCHLESSINGER
• 金额: $ 288.74万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-28 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3106311
• 关键词: artificial chromosomes; fungal genetics; genetic mapping; polymerase chain reaction

Yeast artificial chromosomes (YACs) as a cloning vector provide the basis for an approach to the systematic analysis of the contents of the human genome. YACs can clone much or all of the human genome faithfully in fragments of average size 250 kb or more, and YACs cognate for specific probes can be recovered from libraries and assembled in contigs of average size at least 2 Mb. In work thus far, a collection of YACs containing five genomic equivalents of human DNA has been organized in matrix arrays and screened for cognate YACs in a Core Screening Lab. Screening involves the use of specific primer pairs and the Polymerase Chain Reaction (PCR) on YAC DNAs from pools of colonies, and then detection of individual clones by hybridization of the PCR product as a probe against DNA from the colonies in positive pools. Through the efforts of the Development laboratory in conjunction with Core Units and collaborating laboratories, the P50 Center seeks to increase the efficiency of screening for YACs 3 to 10-fold, and thus to approach three-year goals of 1) screening for up to 1500 YACs covering individual genes and regions of up to 4 Mb as well as 600 YACs specific for chromosome 4 (Project 1); and 2) mapping up to 3000 YACs across each of chromosomes 7 and X (Projects 2 and 3). This throughput could be achieved by screening completely by PCR methods, and by generating primer pairs independent of the YACs themselves. [Primer pairs are to be derived from other venues for other loci; for chromosomes 7 and X, they are to be generated in the STS Core Lab from specialized libraries of 200 to 1000 base pair fragments cloned from flow-sorted chromosomes.] Each primer pair defines a sequence-tagged site [STS, as in Olson et al. (Science 245;1434-1435)] in a cognate YAC. The alignment of YACs, and the organization of the resultant map, is based first on their content of different STS; second, on their relation to the genetic linkage map, as refined in the Linkage Laboratory; and third, on auxiliary physical mapping studies, including the use of jumping clones and in situ hybridization. With the aid of the Information Handling and Analysis Core, the YAC contigs would provide a basis for the merger of genetic and physical markers into a single unified map. The Genome Project relatedness of the Center would thus include provision of about 15% of the unified map, in a format easy to refine, using more efficient and less costly methods than are currently available.

酵母人工染色体（YAC）作为克隆载体提供了基础 寻找一种对人类内容进行系统分析的方法 基因组。 YAC 可以忠实地克隆大部分或全部人类基因组 平均大小为 250 kb 或更大的片段，YAC 与特定的片段同源 探针可以从文库中回收并组装成平均数量的重叠群 大小至少 2 Mb。 迄今为止的工作中，包含五个的 YAC 集合 人类 DNA 的基因组等价物已被组织成矩阵阵列 在核心筛选实验室中筛选同源 YAC。筛查涉及 在 YAC 上使用特定引物对和聚合酶链式反应 (PCR) 从菌落池中提取 DNA，然后通过以下方法检测单个克隆 PCR 产物作为探针与菌落 DNA 杂交 在正池中。 经过研发实验室的努力 与核心单位和合作实验室合作，P50 中心 力求将 YAC 筛选效率提高 3 至 10 倍，并且 从而实现 1) 筛选多达 1500 个 YAC 的三年目标 覆盖高达 4 Mb 的单个基因和区域以及 600 个 YAC 针对 4 号染色体（项目 1）； 2) 映射多达 3000 个 YAC 跨 7 号和 X 号染色体（项目 2 和 3）。 这个吞吐量 可以通过PCR方法完全筛选并生成 引物对独立于 YAC 本身。 [引物对应为 来自其他基因座的其他场所；对于 7 号和 X 号染色体，它们是 将在 STS 核心实验室中从 200 个到 从流式分选染色体克隆的 1000 个碱基对片段。] 每个引物 对定义了一个序列标记位点 [STS，如 Olson 等人中所述。 （科学 245;1434-1435)] 在同源 YAC 中。 YAC 的对齐，以及 结果地图的组织首先基于其内容 不同的STS；其次，关于它们与遗传连锁图的关系，如 在联动实验室精炼；第三，关于辅助物理测绘 研究，包括使用跳跃克隆和原位杂交。 在信息处理和分析核心的帮助下，YAC 重叠群 将为将遗传和物理标记合并为一个基础 单一统一地图。 该中心的基因组计划相关性将 因此，包括以易于理解的格式提供约 15% 的统一地图 使用比目前更有效、成本更低的方法进行精炼 可用的。