TRP ion channel and AChR SNP assays

For each SNP, two PCR primers and one extension primer were created using the Assay Designer (Sequenom) according to the manufacturer’s instructions. A tag was added to the 5′-end of the sequence for each primer. This was done to increase their mass so they were not detected by MassArray when analyzing the extension primers. DNA was amplified via PCR under the following conditions: 94°C for 2 minutes, 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1 minute. Amplification products were then treated with shrimp alkaline phosphatase at 37°C for 40 minutes, 85°C for 5 minutes’ reaction, and a final incubation at 4°C. Extension primers were optimized to control the signal-to-noise ratio, where UEPs are examined on the SpectroChip and evaluated in Typer 4.0 to enable the division into low-mass UEP, medium-mass UEP, and high-mass UEP. To perform the iPlex extension reaction, a mixture containing iPlex Gold reaction was prepared using iPlex Gold Buffer Plus, iPlex termination mix, iPlex enzyme, and primer mix. The iPlex reaction was cycled at an initial denaturation of 94°C for 30 seconds, annealing at 52°C for 5 minutes, extension at 80°C for 5 minutes (five cycles of annealing and extension were performed, but the whole reaction was performed in 40 cycles), and extension again at 72°C for 3 minutes. Resin beads were used to rinse all iPlex Gold reaction products. Following the iPlex Gold reaction, spectrometry was performed using the MassArray mass spectrometer, and the data generated were analyzed using the TyperAnalyzer software.