The Decode Universal Mutation Detection System TM (BioRad, Hercules, CA) was used for the sequence-specific separation of the PCR products. Electrophoresis was performed in a 0.8-mm polyacrylamide gel (8% [wt/vol] acrylamide:bisacrylamide 37.5:1) and a 30–60% urea-formamide denaturing gradient (100% corresponds to 7 M urea and 40% [w/v] formamide). The gel was subjected to a constant voltage of 60 V for 12 h at 60°C. After electrophoresis, the gel was stained for 20 min in a SYBR Green solution (Cambrex, Bio Science Rockland, Inc.) and analyzed under UV illumination (VersaDoc 4000 Imaging System, BioRad)