Bigdye terminator 3.1 ready reaction mix

Following PCR‐RFLP gel electrophoresis, patients’ samples exhibiting anomalous run, compared to normal wild type, were Sanger sequenced in both directions to verify the presence of LRTOMT c.242G>A (p.Arg81Gln) variant. Briefly, PCR products were gel purified using the GeneJet gel extraction kit (Thermo Scientific) and then sequenced on the ABI 3500 Genetic Analyzer (Applied Biosystems) using either reverse or forward primer and 4 µl of BigDye terminator 3.1 ready reaction mix (Applied Biosystems). Sequencing chromatograms were analysed using Chromas software V2.6.4 followed by comparison to NCBI database.

Using the amplified full-length KIT cDNA as a template, exons 9, 11, 13, and 17 were analyzed using the directional sequencing method on an ABI 310 system (Applied Biosystems, Foster City, CA). Sequence primer was shown in Table 1 and Fig. 2). 2 μl of PCR product was mixed with 4 μl of 50 times diluted primer, 1 μl of Big Dye terminator 3.1 Ready Reaction Mix (Applied Biosystems, Foster City, CA), 4 μl of Sequencing buffer and water. A total volume of 20 μl was performed sequencing reaction over 25 cycles of the following step (10 s of denaturation at 96 °C, 5 s of annealing at 50 °C and 1 min of extension at 60 °C). In GISTs which were negative for KIT mutations, exons 12, 14, and 18 of the PDGFRA gene were further analyzed after PCR amplification of full-length PDGFRA cDNA. GIST that did not possess KIT or PDGFRA mutation was defined as wild-type GIST.