MOUSE GENOME MAP WITH BACTERIAL ARTIFICIAL CHROMOSOMES

我们开发了一种基于细菌F因子的系统，用于克隆大鼠 在大肠杆菌(BAC系统)中的哺乳动物DNA片段。的主要目标是 本研究的目的是构建BAC小鼠基因组文库 载体，并使用这些克隆对小鼠基因组进行物理映射。 之前对人类基因组BAC载体的研究表明，体型较大的人类 插件不是嵌合式的，它们是稳定的、具有代表性的 相对容易操纵。 小鼠文库将由排列的细菌克隆组成，平均 插入大小约为175kb，相当于覆盖范围的4-5倍 小鼠的基因组。克隆将被渲染到高密度滤镜上 用于菌落杂交，并将被汇集以允许文库筛选 用聚合酶链式反应。我们将使用多种不同的方法来识别克隆 与已建立的物理和遗传地标相对应。首字母 作图实验将重点放在小鼠染色体上的 充满记号笔的。我们将使用该库来隔离克隆 起源于小鼠17号染色体的近端区域，并建立 “重叠群”，即一组重叠的克隆。我们还将重点关注其他 具有高密度遗传和物理图谱的区域 记号笔。这些实验将为我们提供快速 验证库的代表性程度以及 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，我们还将隔离BAC克隆 其中包含由Eric Lander的团队和其他人开发的STSS 将提供几乎完全覆盖小鼠基因组的小组 在BAC里。我们还将把荧光技术应用于 用原位杂交技术分析小鼠基因组。我们将实施BAC 与中期染色体杂交以定位BAC和确认定位 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.