Mini-dizileme ile Kanserdeki Moleküler Hedeflerin Hassas Tayini

Amaç: Belirli mutasyonlara neden olan moleküler değişiklikler tümör gelişimi ve sağkalımı için gereklidir. Bu moleküler hedeflerin doğru analiz edilmesi farklı kanser tiplerinde tanı, erken teşhis, prognoz tahmini ve tedavide yol gösterme açısından önemlidir. Bu nedenle, moleküler belirteçlerin hassas tayini için Sanger dizilemenin yanı sıra mini-dizileme protokolünü de optimize ederek kullanmayı amaçladık. Yöntem: Gliom örneklerinden elde edilmiş genomik DNAlar kullanılarak IDH1 R132, IDH2 R140/R172 ve TERT promotör C228/C250 mutasyonları için Sanger dizileme ve mini-dizileme yapılmıştır. Mini-dizileme reaksiyonları mutasyon saptama primerleri ile “SnaPshot Multiplex Ready Reaction Mix” kullanılarak gerçekleştirilmiş ve otomatik kapiler elektroforezinde yürütülmüştür. Çoklu eğriler “GeneMapper Software” kullanılarak analiz edilmiştir. Bulgular: Mini-dizileme analizlerinde, yabanıl tip alellere ve farklı mutasyonlara ait eğriler tespit edilmiştir. Yabanıl tip piklerin yanında yer alan eğriler, o noktada varyasyona işaret etmekte ve mutasyon renge göre tanımlanmaktadır. Sonuç: Moleküler belirteçlerin kanserde tayini oldukça önemlidir. Minidizileme yöntemi moleküler hedeflerin belirlenmesinde güvenilir bir yöntemdir.

Sensitive Detection of Molecular Targets in Cancer by Minisequencing

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 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 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 point 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.

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