A Cross-Sectional Study for Evaluation of KRAS and BRAF Mutations by Reverse  Dot Blot, PCR-RFLP, and Allele-Specific PCR Methods Among Patients with Colorectal Cancer

Fatemeh   Sheikhsofla; Behzad   Poopak; Sajjad  Firuzyar; Fatemeh Roudbari; Mojtaba   Ghadiany

doi:10.18502/ajmb.v13i4.7203

Fatemeh Sheikhsofla Department of Cellular and Molecular Biology, University of Mazandaran, Mazandaran, Iran
Behzad Poopak Department of Clinical Laboratory Sciences, Islamic Azad University, Tehran Medical Sciences Branch, Tehran, Iran
Sajjad Firuzyar Razi Vaccine and Serum Research Institute of Karaj, Karaj, Iran
Fatemeh Roudbari Department of Virology, University of Mazandaran, Mazandaran, Iran
Mojtaba Ghadiany Department of Hematology and Oncology, Shahid Beheshti University of Medical Sciences, Tehran, Iran

DOI: https://doi.org/10.18502/ajmb.v13i4.7203

Keywords: Allele-Specific PCR, BRAF, Colorectal neoplasms, KRAS, PCR-RFLP, Reverse dot blot

Abstract

Background: KRAS and BRAF genes are the biomarkers in Colorectal Cancer (CRC) which play prognostic and predictive roles in CRC treatment. Nowadays, the selection of rapid and available methods for studying KRAS and BRAF mutations in anti-EGFR therapy of patients suffering from CRC plays a significant role. In this study, the mutations of these two oncogenes were evaluated by different methods.

Methods: This study was performed on 50 Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks of patients diagnosed with colorectal cancer. After DNA extraction, KRAS and BRAF gene mutations were evaluated using reverse dot blot, and results were compared with PCR-RFLP and allele-specific PCR for KRAS and BRAF mutations, respectively.

Results: KRAS gene mutations were detected in 42% of patients, of which 30% were in codon 12 region, and 12% in codon 13. The most frequent mutations of KRAS were related to G12D and 10% of patients had BRAF mutated genes. The type of KRAS gene mutations could be evaluated by reverse dot blot method. In general, the results of PCR-RFLP and allele-specific PCR were similar to the findings by reverse dot blot method.

Conclusion: These findings suggest that PCR-RFLP and allele-specific PCR methods are suitable for screening the presence of the mutations in KRAS and BRAF oncogenes. In fact, another method with more sensitivity is needed for a more accurate assessment to determine the type of mutations. Due to higher speed of detection, reduced Turnaround Time (TAT), and possible role of some KRAS point mutations in overall survival, reverse dot blot analysis seems to be an optimal method.