Localization of a unique gene by direct hybridization in situ.

Abstract

Previous determinations of the chromosomal location of unique genes have required that the chromosomes of interest be fractionated, either by species-specific chromosome loss from interspecies hybrids, or by physical fractionation procedures. A general technique for the localization of a unique gene, which requires no prior chromosome fractionation, is reported. The technique involves the use of a labeled hybrid c[complementary]DNA plasmid for direct hybridization in situ to metaphase cells from the organism under investigation. As a model system for development of this technique, a human .alpha.-globin cDNA plasmid (JW101) is employed to localize the corresponding gene cluster. To obtain a sufficiently large autoradiographic signal, this plasmid was labeled with 125I to a high specific activity (109 dpm[disintegrations/min]/.mu.g) and the ability of a double-stranded probe to form networks was utilized. To obtain sufficient hybridization specificity, various experimental procedures were used, the most important of which was the choice from among a range of probe concentrations of the highest that does not yield excessive background hybridization. With an autoradiographic exposure time of only 12 days, use of this technique correctly localized the human .alpha.-globin gene cluster to chromosome 16. This technique should be generally applicable to the localization of any gene for which a corresponding cDNA hybrid plasmid is available.