Snapshot multiplex kit system

SNaPshot analysis of SNP genotypes was performed using a SNaPshot Multiplex kit system (Applied Biosystems, Foster City, CA, USA). Products were treated with 1 unit of shrimp alkaline phosphatase at 37°C for 60 min and 75°C for 15 min, followed by a denaturation step at 95°C for 5 min. Detection was performed using 0.5 μl SNaPshot products mixed with 9 μl HiDi™ formamide and 0.5 μl GeneScan-120LIZ size standard (Applied Biosystems). Data were generated following capillary electrophoresis on an automated sequencer (ABI 3130 Genetic Analyzer; Applied Biosystems) with a 36-cm length capillary and POP-7™ polymer and analyzed using GeneMapper^® software version 3.7 (Applied Biosystems).

SNaPshot analysis of SNP genotypes was performed to confirm the remaining variants after variant filtration in the samples of patients and controls, using a SNaPshot Multiplex kit system (Applied Biosystems, Foster City, CA, USA). Specific primers were designed using the Primer 3 Software (Supplemental Table 5). SNaPshot results were analyzed using Gene Mapper v4.1 software (Foster City, CA, USA).