MOUSE GENOME MAP WITH BACTERIAL ARTIFICIAL CHROMOSOMES

我们开发了一种基于细菌 F 因子的系统，用于克隆大 大肠杆菌（BAC 系统）中的哺乳动物 DNA 片段。主要目标 本提案是在 BAC 中构建小鼠基因组文库 载体并使用这些克隆进行小鼠基因组的物理作图。 先前对人类基因组 BAC 载体的研究表明，大型人类 插入片段不是嵌合的，它们是稳定的、有代表性的，并且 相对容易操纵。 小鼠文库将由排列好的细菌克隆组成，平均细菌克隆数为 插入大小约为 175kb，将代表 4-5 倍的覆盖率 小鼠基因组。克隆将被渲染到高密度过滤器上 用于菌落杂交，并将合并以进行文库筛选 通过PCR。我们将使用多种不同的方法来识别克隆 对应于已建立的身体和遗传标志。初始 作图实验将集中于小鼠染色体的区域 充满标记。我们将使用该库来分离克隆 起源于小鼠 17 号染色体的近端区域，并建立 “重叠群”，或重叠克隆组。我们还将关注其他方面 具有高密度遗传和物理映射的区域 标记。这些实验将为我们提供快速 验证图书馆的代表性程度并 演示使用 BAC 生成详细信息的通用程序 基因组中任何区域的图谱。此外，我们将分离 BAC 克隆 其中包含由 Eric Lander 小组和其他人开发的 STS 组将提供几乎完整的小鼠基因组覆盖 在 BAC 中。 我们还将应用荧光技术 原位杂交对小鼠基因组的分析。 我们将进行BAC 与中期染色体杂交以绘制 BAC 图谱并确认图谱 通过其他方法建立的数据。

We have developed a bacterial F-factor based system for cloning large fragments of mammalian DNA in E coli (the BAC system). The primary aims of this proposal are to construct a genomic library of the mouse in the BAC vector and to use these clones for physical mapping of the mouse genome. Previous work with human genome BAC vectors indicates that the large human inserts are not chimeric, they are stable, representative, and are relatively easy to manipulate. The mouse library will consist of arrayed bacterial clones with average insert sizes of approximately 175kb, and will represent 4-5 times coverage of the mouse genome. The clones will be rendered onto high density filters for colony hybridization, and will be pooled to permit library screening by PCR. We will use a number of different approaches to identify clones corresponding to established physical and genetic landmarks. Initial mapping experiments will focus on regions of the mouse chromosome that are replete with markers. We will use the library to isolate clones originating from the proximal region of mouse chromosome 17, and establish "contigs", or groups of overlapping clones. We will also focus on other regions that have high densities of both genetically and physically mapped markers. These experiments will provide us with the opportunity to quickly verify the extent to which the library is representative and to demonstrate generalizable procedures for using BACs to generate detailed maps of any region in the genome. In addition, we will isolate BAC clones which contain the STSs developed by Eric Lander's group and by other groups which will provide virtually complete coverage of the mouse genome in BACs. We will also apply the techniques of fluorescent in situ hybridization to analysis of the mouse genome. We will carry out BAC hybridizations to metaphase chromosomes to map BACs and confirm mapping data established by other approaches.