QUANTITATIVE POLYMERASE CHAIN REACTION-BASED HOMOGENEOUS ASSAY WITH FLUOROGENIC PROBES TO MEASURE C-ERBB-2 ONCOGENE AMPLIFICATION

We describe a PCR-based assay for determining c-erbB-2 oncogene amplif ication in breast cancer in which we use the TaqMan(TM) system. Two fl uorogenic probes anneal to the target between primers for c-erbB-2 and beta-globin genes and contain both a reporter dye (6-carboxy-fluoresc ein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase of the PCR cycle, the 5'-->3' exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quant itative for the amount of PCR product and, under appropriate condition s, for the amount of template. Assay performance showed adequate preci sion and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 0.94, P <0.01). This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional source s of inaccuracy and contamination), and seems suitable for determinati on of c-erbB-2 oncogene amplification in tumor specimens.