HIGH RESOLUTION OF GENE MAPPING BY IN SITU HYBRIDIZATION

通过原位杂交进行高分辨率基因作图

• 批准号: 3333337
• 负责人: DAVID C WARD
• 金额: $ 82.62万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-05-01 至 1993-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3333337
• 关键词: genetic mapping; genome; human tissue; in situ hybridization

The overall objective of this program project is to apply newly emerging technologies to the large scale physical and genetic mapping of the human genome and the generation of ordered sets of cloned DNA spanning megabase regions of the X chromosome. Specific objectives are: 1)to generate a physical linkage map of the human genome (all chromosomes) by precisely localizing about 6000 cloned DNA or cDNA fragments on metaphase chromosomes by multiparameter in situ hybridization using fluorescence digital imaging microscopy. These DNAs will include about 5000 random phage and cosmid clones, 500 clones known to contain a RFLP, 300 Cla/Cla linking clones (with spacing of 5-10 mega bp) as well as several hundred additional Not/Not or Taq/Taq linking library clones. 2)to carry out genetic linkage studies on 100 DNA markers per year by hybridization to CEPH and other large reference family pedigrees and to screen about 200 clones per year from the panel of clones mapped by in situ hybridization for RFLPs and a subset of these for other types of sequence polymorphism. 3)to prepare a complete set of genomic Cla-Cla linking fragments and a complete set of Taq-Taq or BsuE-methylated Not I for the X chromosome. 4)to apply affinity-"fishing"-techniques to the direct isolation of ordered DNA fragments from the X chromosome and to prepare clone sets for each fragment. 5)to prepare a cDNA expression map of the X chromosome using a) in situ hybridization with cDNA clones, b) cross-library screening to identify tissue-specific patterns of expression (cDNA libraries from multiple human tissues have already been prepared) and c) cDNA library screening with selected clones prepared from affinity purified Cla/Cla DNA fragments. 6)to sequence the panel of cDNA clones identified and mapped as part of research objective #5._

该计划项目的总体目标是应用新兴的 大规模的物理和人类基因图谱的技术 基因组和跨越百万碱基的克隆DNA的有序集合的产生 X染色体的区域。具体目标是： 1）生成人类基因组（所有染色体）的物理连锁图 通过精确定位约6000个克隆的DNA或cDNA片段， 中期染色体的多参数原位杂交 荧光数字成像显微术。这些DNA将包括大约5000个 随机噬菌体和粘粒克隆，500已知含有RFLP的克隆，300 Cla/Cla连接克隆（具有5-10兆bp的间距）以及几个 数百个另外的Not/Not或Taq/Taq连接文库克隆。 2）每年对100个DNA标记进行遗传连锁研究， 与CEPH和其它大的参考家系杂交， 每年从原位定位的无性系中筛选约200个无性系 RFLPs杂交和其它类型序列的RFLPs子集 多态 3）制备一套完整的基因组Cla-Cla连接片段和一个 完整的X染色体Taq-Taq或BsuE甲基化Not I。 4）将亲和性-“钓鱼”-技术应用于有序的直接分离 从X染色体的DNA片段，并准备克隆集的每一个 碎片 5）制备X染色体的cDNA表达图谱，使用a）原位 与cDNA克隆杂交，B）交叉文库筛选以鉴定 组织特异性表达模式（来自多个人的cDNA文库 组织已经制备）和c）cDNA文库筛选 从亲和纯化的Cla/Cla DNA片段制备的选定克隆。 6）对鉴定和定位的cDNA克隆组进行测序，作为 研究目标#5。