Moreover, a scale measuring Spirituality is included in the questionnaire, due to its potential importance for the treatment of alcoholics. The Sadness dimension that is relevant to
this study refers to feelings of loneliness and distress, thinking about loved ones and frequent crying. Genotyping DNA was extracted from buccal mucosa cell samples. Automated purification Inhibitors,research,lifescience,medical of genomic DNA was conducted by means of the MagNA Pure LC system using a commercial extraction kit (MagNA Pure LC DNA isolation kit; Roche Diagnostics, Mannheim, Germany). The VNTR polymorphism of the DAT1/SLC6A3 gene (rs#28363170) was amplified from genomic DNA using polymerase chain reaction (PCR) and the primers 5′-TGTGGTGTAGGGAACGGCCTGAG-3′ and 5′-CTTCCTGGAGGTCACGGCTCAAGG-3′. In brief, after an initial denaturation
for 3 min at 94°C, 39 cycles of denaturing at 94°C for 45 sec, annealing at 62°C for 30 sec, and extension at 72°C for 30 sec were followed by a final extension at 72°C for 5 min. PCR amplification was carried out in a final volume of 20 μl consisting of 50 ng genomic DNA, 0.2 mM of each deoxyribonucleotide, Inhibitors,research,lifescience,medical 1 pmol of sense and antisense primers, 1 U of GoTaq-Polymerase Inhibitors,research,lifescience,medical (Promega, Mannheim, Germany), and the enzyme supplier’s buffer. Amplification products were analyzed by 2% agarose gel electrophoresis. The sizes of the 8, 9, 10, and 11 repeats were 360, 400, 440, and 480 bp, respectively. Genotyping of COMT Val158Met SNPs (rs#4680) was performed by real time PCR (RT-PCR) using fluorescence Inhibitors,research,lifescience,medical melting curve detection analysis by means of the Light Cycler System (Roche Diagnostics, Mannheim, Germany). Details of the PCR protocol are described elsewhere (Reuter et al. 2006). The primers and hybridization probes used (TIB MOLBIOL, Berlin, Germany) were as follows: Inhibitors,research,lifescience,medical forward primer: 5′-GGGCCTACTGTGGCTACTCA-3′; reverse primer: 5′-GGCCCTTTTTCCAGGTCTG-3′; sensor hybridization probe: 5′-ATTTCGCTGGCATGAAGGACAAG-fluorescein-3′; anchor hybridization probe: 5′-LCRed640-TGTGCATGCCTGACCCGTTGTCA-phosphate-3′. Statistical analysis We investigated the influence of COMT and DAT1 on ANPS personality dimensions by means
of analysis of Ku-0059436 in vitro variance (ANOVA). On the genotype levels, the independent factors had three levels each (COMT: Val/Val, Val/Met, and Met/Met; DAT1: 9R/9R, 9R/10R, and 10R/10R). For statistical analyses focusing on gene × gene interactions, COMT and ARCHIVES OF INTERNAL MEDICINE DAT1 genotypes were dichotomized to enhance statistical power. Individuals with the COMT Met/Met and Val/Met genotypes were combined (Met+ and Met− for Val/Val) based on findings that the Val/Val genotype is associated with PEM (Reuter and Hennig 2005) and the Met allele with NEM (Enoch et al. 2003). The DAT1 10R/10R and 10R/9R genotypes were dichotomized for the presence (10R+) or absence (10R−) of the DAT1 10R allele as DAT1 expression is higher in the presence of the 10R allele (Fuke et al. 2001; Mill et al. 2002). Age was negatively correlated with Sadness (r = −.