Development of a novel tetra-primer ARMS-PCR for the identification of hotspot mutation BRAF V600E in patients with melanoma and colorectal cancer
Development of a novel tetra-primer ARMS-PCR for the identification of hotspot mutation BRAF V600E in patients with melanoma and colorectal cancer
Mehran Sepehriyan Bahary Nezhad,1,*Ali Nazemi,2
1. Department of Biology, Faculty of Biological Sciences, Islamic Azad University of Tonekabon, Tonekabon, Iran 2. Department of Biology, Faculty of Biological Sciences, Islamic Azad University of Tonekabon, Tonekabon, Iran
Introduction: The BRAF mutations contribute to different types of cancer, and BRAF mutation screening may provide an effective prognosis. Therefore, the present study aimed to develop tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) for the rapid detection of BRAF V600E mutation in patients with melanoma and colorectal cancer (CRC).
Methods: The present study analyzed 10 deoxyribonucleic acid (DNA) samples extracted from patients' tissues were prepared from a laboratory in Tehran province. Five samples were from patients with melanoma, four of which had an unknown genotype, and one sample had a mutant genotype that was used as a positive control. The other 5 samples had an unknown genotype and were related to patients with colorectal cancer. The DNA samples were analyzed using tetra-primer ARMS-PCR for the presence of BRAF V600E mutations. After the determination of the optimal temperature and optimization of tetra-primer ARMS-PCR based on normal DNA, the main reaction was performed in a microtube.
Results: The analysis of PCR sequencing electropherogram confirmed the presence of BRAF V600E mutation (GTG>GAG) at position c.T1799A in the melanoma DNA sample. In addition, two (22.22%) out of nine unknown genotype DNA samples were BRAF V600E positive. In contrast to the negative control, the development of a sharp BRAF V600E band by positive control indicated the proper functioning of the system used in this study.
Conclusion: In summary, the results of the present study demonstrated that the method of tetra-primer ARMS-PCR is an effective approach for the identification of BRAF V600E mutations. meanwhile, developed primers in this study can detect V600A(GTG > GCG) and V600G(GTG > GGG) mutations in addition to V600E(GTG > GAG) mutation due to their novel design.