HIGH RESOLUTION GENE MAPPING BY IN SITU HYBRIDIZATION

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High resolution mapping of single copy genes on mammalian metaphase chromosomes will be accomplished by in situ hybridization and transmission electron microscopy. The proposed technique is based on that developed for polytene chromosome (Wu, M. and Davidson, N. (1981), Proc. Natl. Acad. Sci. 79, 7059-7083). As electron opaque labels colloidal gold spheres with diameters in the range of 25 nm are used. The spheres are coated with a layer of protein to which E. coli single stranded DNA is photochemically cross-linked. Poly (dA) tails are added to the 3 feet OH ends of a fragmented cloned DNA molecule, corresponding to the gene of interest. These probe-dA strands are hybridized in situ to metaphase chromosome spreads. Gold spheres are linked to the hybridized probe-dA stands by A:T base pairing. The sphere positions relative to the chromosome bands can be observed by transmission electron microscopy. The methods shows low background and high resolution. Genes to be mapped include the repeated ribsomal RNA genes and histone genes, globin genes, immunoglobulins, and histocompatibility genes, on the chromosomes of mice and men.