| Autor: |
E, Lyon, A, Millson, M C, Lowery, R, Woods, C T, Wittwer |
| Rok vydání: |
2001 |
| Předmět: |
|
| Zdroj: |
Clinical chemistry. 47(5) |
| ISSN: |
0009-9147 |
| Popis: |
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 CVs20%. 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 in1 h. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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