HIGH RESOLUTION OF GENE MAPPING BY IN SITU HYBRIDIZATION

该计划项目的总体目标是应用新出现的 人类大比例尺物理和基因图谱绘制技术 基因组与跨越巨型数据库的有序克隆DNA集合的产生 X染色体的区域。具体目标包括： 1)生成人类基因组(所有染色体)的物理连锁图谱 通过将大约6000个克隆的DNA或cDNA片段精确定位在 应用多参数原位杂交技术研究中期染色体 荧光数字成像显微镜。这些DNA将包括大约5000个 随机噬菌体和粘粒克隆，500个已知包含RFLP的克隆，300 CLA/CLA链接克隆(间距为5-10兆bp)以及几个 数百个额外的Not/Not或Taq/Taq链接文库克隆。 2)每年对100个DNA标记进行遗传连锁研究 与CEPH和其他大型参考家系的杂交 每年从现场映射的克隆面板中筛选约200个克隆 RFLP及其子集与其他类型序列的杂交 多态。 3)制备一套完整的基因组CLA-CLA连接片段和 X染色体的全套Taq-Taq或BsuE-甲基化Not I。 4)将亲和力--“钓鱼”--技术应用于直接分离有序的 从X染色体上提取DNA片段，并为每个片段准备克隆集 碎片。 5)利用a)原位技术制作X染色体的cDNA表达图谱 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(来自多个人类 组织已经准备好)和c)用 从亲和纯化的CLA/CLA DNA片段中筛选出克隆。 6)对作为以下部分鉴定和定位的一组cdna克隆进行测序 研究目标#5。

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._