Quantification of HER2/neu gene amplification by competitive PCR using fluorescent melting curve analysis

Background: Molecular detection methods for HER2/neu gene amplification inc lude fluorescence in situ hybridization (FISH) and competitive PCR. We desi gned a quantitative PCR system utilizing fluorescent hybridization probes a nd a competitor that differed from the HER2/neu sequence by a single base c hange. Methods: Increasing twofold concentrations of competitor were coamplified w ith DNA from cell lines with various HER2/neu copy numbers at:he HER2/neu l ocus. Competitor DNA was distinguished from the HER2/neu sequence by a fluo rescent hybridization probe and melting curve analysis on a fluorescence-mo nitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate co py number. Results: Real-time monitoring of the PCR reaction showed comparable relativ e areas throughout the log phase and during the PCI: plateau, indicating th at only end-point detection is necessary. The dynamic range was over two lo gs (2000-250000 competitor copies) with CVs < 20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and II, re spectively. Gene amplification was detected in 3 of 13 tumor samples and wa s correlated with conventional real-time PCR and FISH analysis. Conclusion: Use of relative peal; areas allows gene copy numbers to be quan tified against an interval competitive control in <1 h. (C) 2001 American A ssociation for Clinical Chemistry.