DNA was extracted from patients’ lymphocytic cell pellets using DNA isolation and purification kit supplied from Jena Bioscience (Germany). Genomic DNA (500 ng) was bisulfite modified by (EZ DNA MethylationTM Kit (Zymoresearch, Germany). MS-PCR was carried out following the method of Herman et al.16 PCR was performed using primer pairs which target one or more CpG of the core promoters in the sense strand of the 4 genes (TS, TP, DPD and COX2). Two sets of primers for each gene were designed by Meth prime design tool. The primers that amplify sequences in which CpGs are methylated (M-primers), and the primers that amplify sequences in which CpGs are unmethylated (U-primers) were purchased from Invitrogen by ThermoFisher Scientific, UK Table 1. In 25 µl, PCR amplification was carried out using host start Taq DNA polymerase master mix (Zymo research, Germany), bisulfite-modified DNA template (2 µl) and 10 µM of the forward and reverse primers. Amplification conditions was adjusted according to manufacturer recommendations and genes’ annealing temperatures optimised, as seen in Table 1. Control PCRs lacking genomic DNA were performed for each set of reactions. PCR reaction products were loaded onto horizontal electrophoresis on agarose gels, stained with ethidium bromide and visualised under UV illumination.

MS-PCR primers.

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