A total of 58 SNPs mapping within and nearby genes encoding 10AA transporters were prioritized by a tagging approach, attempting to choose those most likely to be of functional relevance (nonsynonymous SNPs, SNPs located in the 5′ and 3′ UTR regions). Supplementary Table S1 reports the complete list of selected SNPs, their position (relative to the chromosome and to the gene), and putative functional annotation.
Multiplex SNP genotyping was performed using PCR followed by primer extension and MALDI-TOF mass spectrometry using iPLEX Gold technology from Sequenom (Sequenom Inc, San Diego, USA). Sequenom MassARRAY Assay Designer software (version 3) was used to design primers for PCR and single base extension. Standard procedures were used to amplify PCR products, and unincorporated nucleotides were deactivated with the shrimp alkaline phosphatase (SAP). A primer extension reaction was implemented by mass extension primer and terminator. The primer extension products were then desalted on resin, and spotted onto the 384-element SpectroCHIP (Sequenom) for MALDI-TOF analysis using Spec-troACQUIRE v3.3.1.3 (Sequenom). Spectra were analyzed using MassARRAY Typer v3.4 Software (Sequenom). Approximately 10% of the samples were analysed in duplicate, and the concordance rate of the genotypes was higher than 99%.
Multiplex SNP genotyping was performed using PCR followed by primer extension and MALDI-TOF mass spectrometry using iPLEX Gold technology from Sequenom (Sequenom Inc, San Diego, USA). Sequenom MassARRAY Assay Designer software (version 3) was used to design primers for PCR and single base extension. Standard procedures were used to amplify PCR products, and unincorporated nucleotides were deactivated with the shrimp alkaline phosphatase (SAP). A primer extension reaction was implemented by mass extension primer and terminator. The primer extension products were then desalted on resin, and spotted onto the 384-element SpectroCHIP (Sequenom) for MALDI-TOF analysis using Spec-troACQUIRE v3.3.1.3 (Sequenom). Spectra were analyzed using MassARRAY Typer v3.4 Software (Sequenom). Approximately 10% of the samples were analysed in duplicate, and the concordance rate of the genotypes was higher than 99%.