Pyromark assay design software 2

Genomic DNA was extracted from peripheral blood using guanidine isothiocyanate and following
phenol-chloroform extraction using a standard protocol (Słomski 2008) . Isolates were dissolved in 1× TE buffer and stored at -20°C until analysis. Then, SNPs in the RET gene were analyzed by pyrosequencing or high-resolution melting analysis (HRMA) and Sanger sequencing. Primers were designed using PyroMark Assay Design Software 2.0 (Biotage) and Primer 3 online primer design tool (all primer sequences and melting temperatures are listed in Supplementary Table 2). Templates for pyrosequencing were amplified by PCR as follows: a 5-min initial denaturation at 94°C, then 50 cycles of 94°C for 30 s, annealing for 30 s and 72°C for 60 s, followed by a 5-min final elongation at 72°C. Pyrosequencing reactions were performed on the PSQ96 apparatus using PyroMark Gold Q96 reagents kits (Qiagen) according to the manufacturer's recommendations. HRM analyses were run on the Rotor-Gene Q cycler using the Type-it HRM PCR Kit (Qiagen). The DNA fragments containing the RET gene exons were amplified by PCR (5 min at 95°C, then 40 cycles as follows: 95°C for 10 s, 60°C for 30 s and 72°C for 10 s) and the products were subsequently melted from 75 to 95°C by raising the temperature by 0.1°C at each step. Sanger sequencing was performed on MegaBACE 500 (GE Healthcare) or ABI PRISM 3100 (Applied Biosystems).