PHYSICAL MAPPING OF THE SHORT ARM OF CHROMOSOME 11

11 号染色体短臂的物理图谱

• 批准号: 3878809
• 负责人: SARASWATI SUKUMAR
• 金额: --
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3878809
• 关键词: centromere; chromosomes; genetic library; genetic manipulation; genetic mapping; genetic markers; genome; human tissue; in situ hybridization; molecular cloning; nucleic acid probes; nucleic acid sequence; restriction mapping; transposon /insertion element; yeasts

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.

这个项目的目标是获得一个高分辨率的物理地图， 11号染色体短臂。 到目前为止，11号染色体和X是最好的 其特征在于，具有最大数量的探针和覆盖的范围， 重叠克隆 因此，这里制定的战略可以作为 这是绘制基因组其他区域图谱的范例。 我们的战略 包括在酵母人工培养系统中制备11号染色体文库， 使用来自流式分选的染色体11的DNA， 含有这条染色体作为其唯一人类材料的体细胞杂交体。 将YAC克隆排列在96孔板中并存档，每个克隆的名称 克隆是根据其网格坐标分配的。 鉴定 携带定位到短臂的基因和标记的YAC克隆， 通过多重程序进行PCR分析/杂交/筛选克隆 将揭示文库中染色体11 p表达的范围。 YAC克隆的人DNA插入片段的存在和大小的表征， 稀有限制性位点的作图， 通过环化的YAC片段的反向PCR，和原位 YAC克隆直接或随后与中期染色体杂交 亚克隆到Cosmos中，将有助于确定 克隆和构建重叠群。 我们需要一套100 YAC 平均插入片段大小为500 kb的克隆和1250个插入片段大小为40 kb的粘粒克隆 插入，端对端对齐，以跨越大约50 mb的短臂， 11号染色体。 基因或基因座的进一步详细分析， 携带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表征。 测序 使用T7或T3启动子位点的双链粘粒DNA模板， 存在于粘粒载体中的序列标记位点（STS）将产生用于 每一个粘粒克隆，一个参考点的进展， 项目，并提供了一个方法，地图关闭。 通过使用 我们将着手进行的YAC克隆和粘粒克隆的组合， 在项目1中生成，我们的目标是提供一个短的物理地图 臂，界标探针的间距为约300 kb。 此外，本发明还提供了一种方法， 该项目将详细研究染色体的一个区域， 11p15.5到pter，具有非常缺乏的标记和定位基因， 随后研究了从着丝粒到11p15.5的区域。 我们希望解开健康和疾病中重要的基因， 11号染色体短臂的详细物理图谱，并产生 DNA测序的模板。