PHYSICAL MAPPING OF THE SHORT ARM OF CHROMOSOME 11

这个项目的目标是获得一张高分辨率的地球物理地图 11号染色体的短臂。到目前为止，11号染色体和X染色体是最好的 具有最大探测数和覆盖的范围 重叠的克隆。因此，这里制定的战略可以作为 一种绘制基因组其他区域的范例。我们的战略 包括在人工酵母中制备11号染色体文库 用Flow分选的11号染色体纯化的DNA进行染色体(YAC) 以该染色体为唯一人类材料的体细胞杂交。 YAC克隆将被排列在96个孔板上并存档，每个克隆的名称 根据其栅格坐标指定的克隆。身份识别 YAC克隆含有定位到短臂的基因和标记，如下 用多重方法进行克隆的PCR分析/杂交/筛选 将揭示11p染色体在文库中的表现范围。 YAC克隆对人DNA插入片段的存在和大小的鉴定， 稀有限制酶切点的作图，序列的鉴定 末端通过环状YAC片段的反向聚合酶链式反应，并原位 YAC无性系与中期染色体的直接杂交或后续杂交 克隆到宇宙中，将有助于确定宇宙的方向 克隆和构建重叠群。我们需要一套100元人民币 平均插入500kb的克隆，40kb的粘粒克隆1250个 插入物，首尾对齐，横跨大约50mb的短臂 11号染色体。进一步的基因或基因座的详细分析，作图 含有潜在疾病基因的区域、它们的克隆和 将在粘粒亚克隆中进行鉴定。按顺序排列 使用T7或T3启动子位点的双链粘粒DNA模板， 存在于粘粒载体中将产生序列标签位点(STS) 每一个宇宙中子体克隆，都是 该项目还提供了一种关闭地图的方法。通过使用 我们将着手的YAC克隆和宇宙克隆的组合 要在项目1中生成，我们的目标是提供一个简短的物理地图 臂，标志性探头的间距约为300 kb。此外, 这个项目将详细地研究染色体的一个区域。 11p15.5到PTER，具有极少的标记和定位基因， 随后对该区域从着丝粒到11p15.5进行了研究。 我们希望解开对健康和疾病很重要的基因，产生一种 11号染色体短臂的详细物理图谱，并制作 DNA测序的模板。

The goal of this project is to obtain a high resolution physical map of the short arm of chromosome 11. To date, chromosome 11 and X are the best characterized, with the maximum number of probes and the extent covered by overlapping clones. As such, the strategies developed here could serve as a paradigm for the mapping other regions of the genome. Our strategy consists of preparing chromosome 11 libraries in yeast artificial chromosomes (YAC) using DNA from flow sorted chromosome 11 purified from somatic cell hybrids containing this chromosome as its only human material. YAC clones will be arrayed in 96-well plates and archived, the name of each clone being assigned according to its grid coordinates. Identification of YAC clones harboring genes and markers mapping to the short arm, following PCR analysis/hybridization/screening of clones by the multiplex procedure will reveal the extent of the chromosome 11p representation in the library. The characterization of YAC clones for presence and size human DNA insert, mapping of rare restriction sites, the identification of sequences at the extreme ends by inverse PCR of circularized YAC fragments, and in situ hybridization of YAC clones to metaphase chromosomes directly or following subcloning into cosmids, will assist in determining orientation of the clones and in construction of contigs. We will need a set of 100 YAC clones with an average of 500 kb inserts, and 1250 cosmid clones with 40 kb inserts, aligned end to end, to span the roughly 50 mb short arm of chromosome 11. Further detailed analysis of genes or loci, mapping of regions harboring potential disease genes, their cloning and characterization will be carried out in cosmid subclones. Sequencing of double stranded cosmid DNA template using the T7 or T3 promoter sites, present in the cosmid vector will generate sequence tagged site (STS) for each of the cosmid clones, a reference point for the progress of the project as well as providing an approach for map closure. By using a combination of YAC cloning which we will embark upon, and the cosmid clones to be generated in Project 1, we aim to provide a physical map of the short arm with spacing of landmark probes at about 300 kb apart. In addition, this project will be investigating, in detail, a region of chromosome 11p15.5 to pter that has a great paucity of markers and localized genes, followed later by studies into the region from the centromere to 11p15.5. We hope to unravel genes important in health and disease, produce a detailed physical map of the short arm of chromosome 11, and produce templates for DNA sequencing.