Competitive-Differential Polymerase Chain Reaction for Gene Dosage Estimation oferbB-1 (egfr),erbB-2, anderbB-3 Oncogenes

摘要

Increases in the average gene copy number (AGCN) of theerbB oncogenes, especially theerbB-2 gene, have been found in a variety of human cancers. Such information is useful with respect to prognosis of the disease. Poor reproducibility and DNA damage complicate quantitative polymerase chain reaction (PCR)-based methods. Therefore, we describe a quantitative PCR method for the estimation of AGCN in the oncogeneserbB-1 (egfr),erbB-2, anderbB-3. The method comprises a competitive and differential PCR in a one-tube reaction (competitive-differential PCR, or cdPCR). Genomic sequences of the oncogene and the human β-globin (HBB) reference gene and two competitors for the oncogene and reference gene were amplified with two primer pairs simultaneously. The competitors were chosen to be amplified with the same efficiency as the genomic sequences. Using this method, we confirmedegfranderbB-2 amplification in cancer cell lines and tumor tissue, and we also detectederbB-3 amplifications. Furthermore, gene dosage decreases were detectable,e.g., anerbB-2 hemizygosity in MCF-7 cells. Thus, cdPCR facilitates the detection of both increases and decreases in AGCN with high reproducibility, sensitivity, and accuracy. This method is therefore suitable for clinical studies onerbB oncogene dosage deviations