Gene amplification for c-erbB-2, c-myc, epidermal growth factor receptor, int-2, and N-myc measured by quantitative PCR with a multiple competitor template

Abstract

We recently proposed a quantitative PCR procedure for the absolute measurement of c-erbB-2 oncogene amplification, based on the simultaneous polymerase chain reaction (PCR) amplification of the target gene and of a competitor DNA molecule acting as internal standard. To increase the number of assayable oncogenes and the accuracy of the quantitative comparison of gene amplification degree within the same tumor, we have now constructed a single synthetic competitor for int-2, c-myc, N-myc, epidermal growth factor receptor, and c-erbB-2 genes, and for the reference gene β-globin. This competitor was constructed by a two-step recombinant PCR procedure and inserted as a 297-bp sequence in a plasmid. The order of primer insertion was designed to obtain competitors of comparable sizes to those of the respective genomic targets, but still easily recognizable from the latter ones by gel electrophoresis. The cloned competitor was tested to evaluate the linearity range for each assay. The present application of quantitative PCR based on a multiple competitor represents the first approach for the achievement of a single reagent for the evaluation of a panel of genes potentially amplified in human tumors.