Design and characterization of a SYBR Green I-based melting curve method for investigation of HER2I655V polymorphism in breast cancer

Abstract

Abstract Background Breast cancer is a disease in which cell grows rapidly forming a mass in the breast. HER2 polymorphisms Ile655Val have been studied as biomarkers for breast cancer and may comprise a risk factor of cardiac toxicity for breast cancer-consuming trastuzumab. Aim of work: In this study, we developed a simple, low cost, and rapid test to detect polymorphism at HER2 gene using SYBR Green I-based melting curve method. Subjects and methods In this report, we performed allelic discrimination with real-time temperature melting (Tm) Shift SYBR Green I-based melting curve method. The melting profiles of amplified DNA HER-2 Ile655Val and its characteristics were analyzed. Result Tm value of HER2 GG and AA alleles were 85 ± 0.14 °C and 82.5 ± 0.23 °C, respectively, while cycle threshold (Ct) value of GG, AG, and AA alleles were 19.6 ± 0.27, 22.5 ± 0.23, 18.6 ± 0.22 correspondingly; furthermore, no template control has shown consisting Ct value at 31.18 ± 0.27. The developed methods’ characteristics were optimum annealing at 62 °C and Kappa coefficient value 1 with the mean almost consistent with PCR-sequencing. The coefficient of variability for intra-assay of GG, AG, and AA was in the range of 0.2–1%, while the coefficient of variability for inter-assay for each were in the range 0.7–1%. Further, based on PCR, shelf-life assay has shown stability for 3 months of storage observation. Conclusion This approach may be considered as simple, rapid, and low cost supporting the rapid study of HER2 epidemiology. Furthermore, the developed methods potentially facilitate clinicians in dealing with breast cancer patients, especially in considering about the cardiotoxicity effect of trastuzumab.

Keywords

• Tm Shift SYBR Green I
• qPCR assay
• HER-2 Ile655Val Detection
• Breast cancer
• Trastuzumab