2.6. Molecular Analyses
DNA was extracted from saliva samples using the QIAamp® DNA Mini Kit (QIAGEN S.r.l., Milan, Italy) according to the manufacturer’s instructions. Purified DNA concentration was estimated by measuring the optical density at 260 nm with an Agilent Cary 60 UV-Vis Spectrophotometer (Agilent technologies Australia (M) Pty Ltd., Victoria, Australia).
Subjects were genotyped for three SNPs at base pairs 145 (C/G), 785 (C/T), and 886 (G/A) of the TAS2R38 locus, which gives rise to three non-synonymous coding exchanges (proline to alanine at residue 49, alanine to valine at residue 262 and valine to isoleucine at residue 296), resulting in the two major haplotypes, PAV (the dominant taster variant) and AVI (the non-taster recessive one) and three rare haplotypes (AAI, AAV, and PVI). The polymerase chain reaction was employed to amplify the short region of the TAS2R38 locus, including the first polymorphisms of interest (rs713598); a 221 bp fragment was amplified with forward 5′CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG-3′ and reverse 5′-AGGTTGGCTTGGTTTGCAATCATC-3′ primers. Amplified samples were digested with HaeIII, according to our previous work [57]. For the rs1726866 and rs10246939 SNPs, TaqMan® SNP Genotyping Assay (C_9506827_10 for the rs1726866 assay and C_9506826_10 for the rs10246939 assay; Applied Biosystems by Life-Technologies Italia, Europe BV) was used [80,81,82] according to the manufacturer’s specifications. Replicates and positive and negative controls were included in all reactions.
Molecular analysis identified nine subjects who were PAV homozygous for TAS2R38 locus, 31 who were heterozygous, and 24 who were AVI homozygous. Three subjects with a rare haplotype of TAS2R38 were excluded.