BACKGROUND: Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change. METHODS: Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number. RESULTS: Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000-250 000 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 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis. CONCLUSION: Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.
BACKGROUND: Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change. METHODS: Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number. RESULTS: Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000-250 000 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 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis. CONCLUSION: Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.
Authors: Kristy A Warner; Erin L Crawford; Aiman Zaher; Robert J Coombs; Haitham Elsamaloty; Stacie L Roshong-Denk; Imran Sharief; Guillermo V Amurao; Yongsook Yoon; Amro Y Al-Astal; Ragheb A Assaly; Dawn-Alita R Hernandez; Timothy G Graves; Charles R Knight; Michael W Harr; Todd B Sheridan; Jeffrey P DeMuth; Robert J Zahorchak; Jeffrey R Hammersley; Dan E Olson; Samuel J Durham; James C Willey Journal: J Mol Diagn Date: 2003-08 Impact factor: 5.568
Authors: Alexandra S Whale; Jim F Huggett; Simon Cowen; Valerie Speirs; Jacqui Shaw; Stephen Ellison; Carole A Foy; Daniel J Scott Journal: Nucleic Acids Res Date: 2012-02-28 Impact factor: 16.971