Quantification of oncogene dosage in tumours by simultaneous dual-label hybridization.
Stickland JE., Tomlinson IP., Ramshaw AL., Bromley L., Potter CG., McGee JO.
Gene amplification and allele loss occur in a variety of human tumours and some have prognostic value. Therefore, techniques which facilitate detection and quantification of gene dosage could have wide applicability in cancer research. Using the INT-2 gene as a model system, a quantitative procedure has been developed for measuring gene copy number using dual-label hybridization to DNA dot blots. A probe specific for the INT-2 gene was labelled with [alpha-32P]dCTP and a probe to beta-actin, the control locus, was labelled with [alpha-35S]dATP. Flat-bed scintillation counting was used to detect and separate the emissions resulting from each bound probe, and gene dosage was calculated from the ratio of INT-2 to the beta-actin probe compared with the ratio derived from constitutional DNA. Calculated ratios of greater than 1.22 and less than 0.78 indicated gene amplification and allelic loss respectively, at the 99% confidence limit derived from the population of 35 constitutional DNAs. The results were validated by RFLP analysis. It is expected that this technique will permit precise gene dosage quantification in many areas.