MICRODISSECTION AND CDNA ISOLATION OF CHROMOSOME 21

21 号染色体的显微切割和 CDNA 分离

• 批准号: 3757056
• 负责人: FA-TEN KAO
• 金额: --
• 依托单位: ELEANOR ROOSEVELT INST FOR CANCER RES
• 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/3757056
• 关键词: Downs syndrome; artificial chromosomes; chromosomes; 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.

该分项目的总体目标涉及使用显微解剖技术， 微克隆技术构建区域特异性显微切割 用于唐氏症高分辨率分子分析的21号染色体文库 综合征表型和分离表达的基因序列 21号染色体。 这些研究将提供直接联系， 21号染色体的细胞遗传学和分子水平的分辨率， 应该有助于寻找负责特定成分的基因 唐氏综合征（Down Syndrome，DS） 具体目标是：（1） 21号染色体亚区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 一种显微切割和微克隆技术， 在这个计划项目下开发的将用于建造图书馆 21号染色体区域包括21 pter-cen、21 cen-q21和21 q22。 特殊的细菌宿主菌株将用于更完整的 在文库中的基因组序列的表示，特别是对于 近端长臂区21 q21，探针在此缺失。 独特 从这些文库中分离序列微克隆， 其特征在于用于分离相应的具有大的 插入物。 这些21号染色体区域特异性文库也将用于 用于核型正常的DS的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cDNA文库的直接筛选和 固定化滤膜杂交程序。 大规模分离砷 将进行尽可能多的来自21号染色体的cDNA克隆， 分离的cDNA克隆将被表征，区域分配， 测序 将研究这些区域特异性cDNA克隆， 参与部分三体的DS个体，以确定可能的 与DS特定组分表型的因果关系。