Sensitive Detection of Molecular Targets in Cancer by Minisequencing

Abstract

ABSTRACT Purpose: Molecular alterations leading to specific mutations are essential for tumor development and survival. Accurate analysis of these molecular targets is important for diagnosis, early detection, forecasting of prognosis and aiding in the treatment of different cancer types. Therefore, for sensitive analysis of molecular markers, we aimed to optimize and use minisequencing protocols besides Sanger sequencing. Methods and Materials: Sanger sequencing and minisequencing were performed for IDH1 R132, IDH2 R140/R172 and TERT promoter C228/C250 mutations using genomic DNA isolated from glioma samples. Minisequencing reactions were performed with detection primers using SnaPshot Multiplex Ready Reaction Mix and run on an automated capillary electrophoresis. Multiplex peaks were analyzed with GeneMapper Software. Results: In the multiplex minisequencing analyses, peaks corresponding to wild type alleles and different mutations were detected. The presence of the peaks next to the wild type peaks points to the presence of variations in that location and the nature of the mutation can be identified according to the color. Conclusions: Identification of molecular markers in cancer is very important. Minisequencing is a reliable method for the detection of molecular targets.