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Physical mapping of genes in somatic cell radiation hybrids by comparative genomic hybridization to cDNA microarrays

2002; BioMed Central; Volume: 3; Issue: 6 Linguagem: Inglês

10.1186/gb-2002-3-6-research0026

1474-760X

Johann Y-D. Lin, Jonathan R. Pollack, Fan-Li Chou, Christian A. Rees, Allen T. Christian, Joel S. Bedford, Patrick O. Brown, Mark H. Ginsberg,

Molecular Biology Techniques and Applications

Somatic cell mutants can be informative in the analysis of a wide variety of cellular processes. The use of map-based positional cloning strategies in somatic cell hybrids to analyze genes responsible for recessive mutant phenotypes is often tedious, however, and remains a major obstacle in somatic cell genetics. To fulfill the need for more efficient gene mapping in somatic cell mutants, we have developed a new DNA microarray comparative genomic hybridization (array-CGH) method that can rapidly and efficiently map the physical location of genes complementing somatic cell mutants to a small candidate genomic region. Here we report experiments that establish the validity and efficacy of the methodology. CHO cells deficient for hypoxanthine:guanine phosphoribosyl transferase (HPRT) were fused with irradiated normal human fibroblasts and subjected to HAT selection. Cy5-labeled genomic DNA from the surviving hybrids containing the HPRT gene was mixed with Cy3-labeled genomic DNA from normal CHO cells and hybridized to a microarray containing 40,185 cDNAs, representing 29,399 genes (UniGene clusters). The DNA spots with the highest Cy5:Cy3 fluorescence ratios corresponded to a group of genes mapping within a 1 Mb interval centered near position 142.7 Mb on the X chromosome, the genomic location of HPRT. The results indicate that our physical mapping method based on radiation hybrids and array-CGH should significantly enhance the speed and efficiency of positional cloning in somatic cell genetics.