Single base extension reactions (SNaPshot Multiplex Kit, Applied Biosystems) were performed in a volume of 10 μl containing: 5 μl of SNaPshot Mtiplex Ready Mix, 3 μl of SAP/ExoI treated PCR products, 1 μl of 0.2 μM SNaPshot probe, and 1 μl of deionized water. The reactions were carried out in a thermocyler (25 cycles of 96°C for 10 seconds, 50°C for 5 seconds, 60°C for 30 seconds). After extension, the products were treated with 1 unit of SAP at 37°C, followed by 15 minutes at 75°C. Samples were mixed with the GeneScan-120 LIZ size standard and analyzed on the 3130 Genetic Analyzer (Applied Biosystems). We used GeneMapper 3.5 software to analyze the results.
