A polymerase chain reaction-based assay for the rapid detection of gene amplification in human tumors

Abstract

A differential polymerase chain reaction (PCR) protocol was established for semiquantitative, nonradioactive detection of gene amplification using a DNA sequencer. Oncogene fragments and control DNA sequences were simultaneously PCR-amplified using fluorescent-labelled primers. Analysis of the PCR products allowed quantitative assessment of gene copy numbers in this coamplification assay. Using this approach, we examined a series of 132 brain tumors for amplification of the epidermal growth factor receptor (EGFR) gene. The same set of tumors was also analyzed by Southern blotting and hybridization with a radiolabelled EGFR probe. Both methods yielded virtually identical results. This technique has a great potential for nonradioactive screening of large tumor panels of oncogene amplification.