Optimization of High Resolution Melting Analysis for Detection of KRAS Gene Mutations

Abstract: Background: Mutations of the KRAS oncogene occur in a variety types of human tumors. By assessing the mutation status of KRAS, clinicians can predict patient response to anti-EGFR therapy such as cetuximab (Erbitux®) or panitumumab (Vectibix®) in patients with metastatic colorectal cancer. The aim of this study was to optimize a real-time PCR method followed by high resolution melting analysis (HRM) in a single step for detection of most common mutations within the KRAS gene. Methods: Seven DNA samples with predefined KRAS mutations and 19 tumor samples from patients with metastatic colorectal cancer were used. KRAS mutation detection was performed by direct sequencing as well as HRM. Optimization was performed using touchdown PCR and co-amplification at lower denaturation-temperature PCR. Results: All DNA samples were successfully analyzed with direct sequencing and HRM. Moreover, the improved amplification efficiency and sensitivity was achieved using optimized PCR run protocol. Conclusion: HRM is a simple, inexpensive and reliable method for mutation detection within KRAS. By applying HRM as prescreening method would help reduce labour, time and costs.