YEAST ARTIFICIAL CHROMOSOME-BASED GENOME MAPPING

基于酵母人工染色体的基因组作图

• 批准号: 3106310
• 负责人: DAVID SCHLESSINGER
• 金额: $ 340.7万
• 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 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3106310
• 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（YACs）作为克隆载体提供了基础 对于人类的内容进行系统分析的方法， 基因组 YACs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，并且YAC与特异性 探针可以从文库中回收并组装成平均 至少2 Mb。 在迄今为止的工作中，一个包含五个 人类DNA的基因组等同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 使用特异性引物对和聚合酶链反应（PCR）对YAC 从菌落池中提取DNA，然后通过 PCR产物作为探针与菌落DNA杂交 在积极的游泳池。 通过开发实验室的努力， 与核心单位和合作实验室，P50中心 寻求将筛选YAC的效率提高3至10倍， 从而实现以下三年目标：1）筛选多达1500个YAC 覆盖单个基因和高达4 Mb的区域以及600个YAC 4号染色体特异性（项目1）;和2）绘制多达3000个YAC的图谱 在7号和X号染色体上（项目2和3）。 该吞吐量 可以通过PCR方法完全筛选， 引物对独立于YAC本身。 [引物对应 来自其他位点的其他场所;对于染色体7和X，它们是 将在STS核心实验室从200个专门的库中生成， 从流式分选的染色体克隆的1000个碱基对片段。 每个引物 对定义了序列标记的位点[STS，如Olson等（Science 245;1434-1435）]在同源YAC。 青年活动中心的定位，以及 结果地图的组织，首先基于其内容， 不同的STS;第二，它们与遗传连锁图的关系， 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 将提供一个基础，为合并遗传和物理标记， 统一的地图。 该中心的基因组计划相关性将 因此，包括提供约15%的统一地图，其格式易于 使用比目前更有效、成本更低的方法进行改进， available.