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


CONCLUSIONS
Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.
Lyon
E
E
Department of Pathology, School of Medicine, University of Utah, Salt Lake 84132, USA. lyone@aruolab.com
Millson
A
A
Lowery
M C
MC
Woods
R
R
Wittwer
C T
CT
D004247:DNA / Q000032:analysis; D005456:Fluorescent Dyes; D018628:Gene Dosage; D018734:Genes, erbB-2; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D016133:Polymerase Chain Reaction / Q000379:methods; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D004247:DNA