Visual demonstration of the organization of the human complement C4 and 21-hydroxylase genes by high-resolution fluorescence in situ hybridization

We analyzed the gene organization in the complement component C4 and 21-hydroxylase (21OH) gene region of the human major histocompatibility complex using visual mapping of stretched DNA by multicolor fluorescence in situ hybridization (FISH), Normally, this region contains a duplicated 21OH-C4 gene cluster (21OHB-C4B-210HA-C4A). Duplication and deletion of one or more copies of the 21OH-C4 gene unit are known to occur frequently. Biotin-labeled cDNA of the C4 gene and digoxigenin-labeled cDNA of the 21OH gene were hybridized to decondensed nuclei of peripheral blood lymphocytes obtained from individuals with various 21OH-C4 haplotypes. Hybridization signals of the C4 and 21OH probes were detected with fluorescein isothiocyanate (green) and rhodamine (red), respectively, Two linear green and rad signal clusters were observed in each nucleus showing the normal haplotype. Gene duplication and deletion were visualized as addition and deletion of the signal cluster, respectively. The DNA types of the 21OH-C4 region determined by FISH were concordant with the results previously obtained by conventional molecular studies. Our high-resolution FISH technique is found to be useful for screening gene duplications and deletions. (C) 1996 Academic Press, Inc.