Each real-time PCR run included within-plate duplicates and an RNase P reference assay. CopyCaller v1.0 software (Applied Biosystems) was used to interpret copy number results. Individual genotypes at SNPs rs1137115 and rs28399435 had been previously determined using a custom designed array as part of a larger study[1], and were found in accord with further Sanger sequencing. Further sequencing was attempted in all subjects using primers previously reported to cover all CYP2A6 exons, as well as 2kb of the 5′ flanking region[24]. SNPs rs4803381 (−1013A/G), rs61663607 (−745A/G), and rs28399433 (−48T/G) were genotyped exclusively by Sanger sequencing. PCR assays for the *12 deletion and *1B 3′ UTR conversion also followed Haberl et al. [24]. All fragments to be sequenced were amplified under the same conditions (25μl volume containing 5x GoTaq Flexi buffer (Promega), 5x Q-Solution (Qiagen), 100μM each dNTP, 400 nM each primer, 1.25 GoTaq Polymerase (Promega); PCR profile: 94°C followed by 34 cycles of 45s at 94°C, 45s at 62° and 1 min at 72°C). DNA sequence was analyzed using Sequencher software (v4.7, Gene Codes Corp). All polymorphisms genotyped were in Hardy-Weinberg equilibrium.
