Optimized dynazyme buffer

DNA was extracted from whole blood samples or from leukocyte enriched buffy coats. Extractions were performed using FlexiGene DNA kit (Qiagen, Hilden, Germany). All coding exons and exon-intron boundaries of both CCR9 and CCL25 were amplified with PCR using primer pairs represented in Supplementary Table 1. The PCR reactions (20 μL) contained 10 mM Tris-HCl (pH 8.8 at 25 °C), 1.5 mM MgCl₂, 50 mM KCl, 0.1% Triton X-100 in 10X Optimized DyNAzyme Buffer (Thermo Fisher Scientific, Waltham, MA, USA), 0.5 mM dNTPs, 1.0 μM of each primer, 80 ng genomic DNA and 1.0 U DyNAzyme II DNA Polymerase (Thermo Fisher Scientific). PCR conditions included initial denaturation at 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 55–60 °C (depending on the PCR product) for 40 s and 72 °C for 1 min. The final extension was performed at 72 °C for 5 min. Successful PCR amplification was confirmed by analyzing PCR products with agarose gel electrophoresis and by UV–Vis spectrophotometry (NanoDrop, Thermo Fisher Scientific). Sequencing was performed by Macrogen Europe B.V. (Amsterdam, the Netherlands) exploiting ABI 3730 (Applied Biosystems, Thermo Fisher Scientific). The sequences were analyzed for SNPs using Sequencher 4.10.1 software (Gene Codes Corporation, Ann Arbor, MI, USA). Sequence data was analyzed further by calculating allele frequencies for found SNPs in the study material.