YEAST ARTIFICIAL CHROMOSOME-BASED GENOME MAPPING

酵母人工染色体(YACs)作为克隆载体提供了基础 对于人类内容的系统分析的一种方法 基因组。YAC可以忠实地克隆许多或全部人类基因组 平均大小为250 kb或更大的片段，以及特定的YAC同源片段 可以从文库中回收探针，并将其组装成平均重叠群 大小至少为2 Mb。到目前为止，工作中的YAC集合包含五个 人类DNA的基因组等价物已经被组织在矩阵阵列中，并且 在核心筛选实验室中筛选同源YAC。筛选涉及到 YAC特异引物的利用及聚合酶链式反应 从菌落池中提取DNA，然后通过 聚合酶链式反应产物作为针对菌落DNA的探针杂交 在正数池中。通过开发实验室的努力， 与核心单位和协作实验室、P50中心合作 旨在将YAC的筛查效率提高3至10倍，以及 因此，要接近1)筛选最多1500名YAC的三年目标 覆盖单个基因和高达4Mb的区域以及600个YAC 特定于4号染色体(项目1)；和2)定位多达3000个YAC 横跨7号和X号染色体(项目2和3)。这一吞吐量 可以通过用聚合酶链式反应方法完全筛选，并通过生成 引物对独立于YAC本身。[引子对将是 来自其他地方的其他基因座；对于7号染色体和X染色体，它们是 将在STS核心实验室从200到200个专业图书馆中生成 从流动分选染色体中克隆的1000个碱基对片段。]每种底漆 Pair定义了序列标记的位点[STS，如Olson等人所述。(科学 245；1434-1435)]在同源YAC中。YAC的排列，以及 结果地图的组织，首先基于它们的内容 不同的STS；第二，它们与遗传连锁图谱的关系，如 在Linkage实验室改进；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 将为将遗传标记和物理标记合并为 单一统一地图。该中心的基因组计划关联性将 因此包括以易于使用的格式提供约15%的统一地图 改进，使用比当前更高效、成本更低的方法 可用。

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.