HIGH RESOLUTION OF GENE MAPPING BY IN SITU HYBRIDIZATION

点击翻译按钮获取中文摘要

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._