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 对于对人类内容的系统分析的方法 基因组。 YAC可以忠实地克隆人类的基因组 平均尺寸为250 kb或更多的片段，YACS的特定片段 可以从库中回收探针，并在平均值的重叠中组装 尺寸至少2 MB。 到目前为止，在工作中，包含五个的YAC集合 人DNA的基因组等效物已在基质阵列中组织，并且 在核心筛选实验室中筛选了同源YAC。筛选涉及 在YAC上使用特定底漆对和聚合酶链反应（PCR） 来自殖民地池的DNA，然后通过 PCR产物的杂交作为对菌落的DNA的探针 在积极的池中。 通过开发实验室的努力 P50中心与核心单位和合作实验室的结合 试图提高YAC 3至10倍筛查的效率，并 因此，要实现三年目标1）筛选多达1500 YACS 覆盖最多4 MB的单个基因和区域以及600 YAC 特定于4号染色体（项目1）； 2）最多绘制3000 YACS 在每个染色体7和X中的每个（项目2和3）中。 此吞吐量 可以通过PCR方法完全筛选，并通过生成 底漆对独立于YAC本身。 [底漆对是 来自其他基因座的其他场所；对于染色体7和X，它们是 从200至200库中的STS核心实验室生成 1000个碱基对片段从流量排序的染色体克隆。]每个底漆 对定义了一个序列标记的位点[STS，如Olson等人。 （科学 245; 1434-1435）] Yacs的一致性和 结果地图的组织首先基于其内容 不同的ST；其次，在与遗传连锁图的关系中 在连锁实验室中精炼；第三，在辅助物理映射上 研究，包括使用跳跃克隆和原位杂交。 借助信息处理和分析核心，YAC重叠群 将为合并遗传和物理标记的基础 单个统一地图。 中心的基因组项目相关性 因此，包括提供约15％的统一地图的提供，以易于 优化，使用比目前更高效且成本更低的方法 可用的。