Buffer g

Genomic DNA was extracted either by 0.5 mL of blood or by two buccal swabs using the QIAamp^® DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Two 60 μL DNA elutions were performed.
Collected DNA was checked by agarose gel electrophoresis, spectrophotometrically quantified and stored at −20 °C. UV spectrophotometric quantification was performed with a SmartSpec™ Plus spectrophotometer (Bio-Rad, Hercules, CA, USA).
The enzymatic digestion of genomic DNA was carried out using SspI restriction enzyme (Thermo Fisher Scientific, Waltham, MA, USA) in order to simulate ccfDNA. It was chosen because it is able to recognize and cut two regions of the β globin gene, upstream of the promoter and inside the second intron, respectively, leaving the region around the mutation intact to avoid interference in primers and probe hybridization. Each reaction had a final volume of 20 μL, containing 50 ng of genomic DNA, 1 U of SspI enzyme and 1× buffer G (Thermo Fisher Scientific). The reaction was incubated at 37 °C for 16 h and finally the enzyme was inactivated at 65 °C for 20 min.

The CCL2 polymorphism (rs1024611) was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using forward and reverse primers (Table 1). The reaction was performed in a total volume of 15 μl, containing 1 μl genomic DNA (60–80 ng/μl), 7.5 μl 1× Master Mix PCR (Ampliqon, Denmark), and 0.3 μM of each outer primer. PCR was performed by denaturing the samples at 95 °C for 3 min followed by 40 cycles including 95 °C for 15 s, 64 °C for 20 s, 72 °C for 25 s, and final extension at 72 °C for 6 min. For RFLP analysis, the PCR product was digested with 10 U/ μl PVUП and 10 x buffer G (Thermo Scientific, Massachusetts, United States) and incubated at 37 °C for 16 h. The digested PCR products were separated on a 2% agarose gel.

The PCR product (10 μL) was digested with ten units of Bcll (for CYP2C8*2) in Buffer G (Thermo Scientific) at 55°C for three hours or five units of BseRI (for CYP2C8*3) in CutSmart Buffer (New England Biolabs) at 37°C for 15 min. Digestion was followed by thermal inactivation of BcII at 80°C for 20 min. Five μL of inactivated digested product mixed with a drop of loading dye was analysed by electrophoresis on a 2.5% agarose gel containing ethidium bromide and visualized under UV light illuminator. The mutant variant of CYP2C8*2 was undigested and observed as a band of 107 bp, while wild type DNA was observed as bands of 57 and 50 bp after Bcll digestion. Results with bands at 107, 57 and 50 bp were recorded as heterozygotes. For CYP2C8*3, wild type DNA was observed as bands of 187 and 282 bp after BseRI digestion. Mutants were undigested with one band at 439 bp and heterozygotes yielded bands of 157,282 and 439 bp.