Quantification of HER2/neu Gene Amplification by Competitive PCR Using Fluorescent Melting Curve Analysis

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. Incre...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 2001-05, Vol.47 (5), p.844-851
Main Authors: Lyon, Elaine, Millson, Alison, Lowery, Mary C, Woods, Rachel, Wittwer, Carl T
Format: Article
Language:English
Subjects:
Biological and medical sciences
DNA - analysis
Fluorescent Dyes
Gene Dosage
Genes, erbB-2
Genital system. Mammary gland
Humans
In Situ Hybridization, Fluorescence
Investigative techniques, diagnostic techniques (general aspects)
Medical sciences
Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques
Polymerase Chain Reaction - methods
Tumor Cells, Cultured
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Summary: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. 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. 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. Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.
ISSN:0009-9147
1530-8561
DOI:10.1093/clinchem/47.5.844