MICRODISSECTION AND CDNA ISOLATION OF CHROMOSOME 21

21 号染色体的显微切割和 CDNA 分离

• 批准号: 5212547
• 负责人: FA-TEN KAO
• 金额: --
• 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/5212547
• 关键词: Downs syndrome; artificial chromosomes; chromosome 21; complementary DNA; gene expression; genetic library; genetic manipulation; human genetic material tag; molecular cloning; molecular pathology; nucleic acid sequence; polymerase chain reaction; trisomy

The overall aims of the subproject involve the use of microdissection and microcloning techniques to construct region-specific microdissection libraries of chromosome 21 for high resolution molecular analysis of Down syndrome phenotypes and for isolation of expressed gene sequences from chromosome 21. These studies will provide direct link between cytogenetic and molecular levels of resolution of chromosome 21 and should facilitate search for genes responsible for specific component phenotypes of Down syndrome (DS). The specific objectives are: (1) Construction of sub-region microdissection libraries of chromosome 21 for isolation and characterization of region-specific microclones, and (2) isolation and characterization of region-specific expressed sequences from chromosome 21. A microdissection and microcloning technology developed under this program project will be used to construct libraries for chromosome 21 regions including 21pter-cen, 21cen-q21 and 21q22. Special bacterial host strains will be used for a more complete representation of genomic sequences in the library, particularly for the proximal long arm region 21q21 where probes are deficient. Unique sequence microclones will be isolated from these libraries and characterized for use in isolation of corresponding YAC clones with large inserts. These chromosome 21 region-specific libraries will also be used for the FISH painting analysis of DS with apparently normal karyotype. The unique sequence microclones from chromosome 21 will be used for isolation of cDNA clones by direct screening of cDNA libraries and by the immobilized filter hybridization procedure. Large scale isolation of as many cDNA clones as possible from chromosome 21 will be carried out and the isolated cDNA clones will be characterized, regionally assigned and sequenced. These region-specific cDNA clones will be studied for involvement in DS individuals with partial trisomy to determine possible causal relationships with specific component phenotypes of DS.

该子项目的总体目标包括使用显微解剖和 构建区域特异性显微切割的微克隆技术 用于唐氏体高分辨率分子分析的 21 号染色体文库 综合征表型和表达基因序列的分离 21号染色体。这些研究将提供两者之间的直接联系 21 号染色体的细胞遗传学和分子水平分辨率 应有助于寻找负责特定成分的基因 唐氏综合症（DS）的表型。 具体目标是：（1） 21号染色体亚区域显微切割文库的构建 区域特异性微克隆的分离和表征，以及（2） 区域特异性表达序列的分离和表征 来自 21 号染色体。显微切割和微克隆技术 该计划项目下开发的项目将用于构建图书馆 对于 21 号染色体区域，包括 21pter-cen、21cen-q21 和 21q22。 特殊的细菌宿主菌株将用于更完整的 文库中基因组序列的表示，特别是对于 近端长臂区域 21q21，其中探针有缺陷。 独特的 序列微克隆将从这些文库中分离出来 特征在于用于分离具有大的相应 YAC 克隆 插入。 这些 21 号染色体区域特异性文库也将被使用 对具有明显正常核型的DS进行FISH染色分析。 来自 21 号染色体的独特序列微克隆将用于 通过直接筛选 cDNA 文库并通过 固定化滤膜杂交程序。 大规模分离as 将从 21 号染色体上克隆尽可能多的 cDNA， 分离的 cDNA 克隆将被表征、区域分配和 已测序。 这些区域特异性 cDNA 克隆将被研究 参与具有部分三体性的 DS 个体以确定可能的 与 DS 特定成分表型的因果关系。