EpiTect PCR Control DNA Set (100)

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ORNi-PCR With Plasmid DNA or Bisulfite-Treated DNA: Plasmids pMD20_p16_M and pMD20_p16_U were used as templates for ORNi-PCR. Alternatively, EpiTect PCR Control DNA Set (QIAGEN, Valencia, CA, USA), bisulfite-treated 293T gDNA, and bisulfite-treated HCT116 gDNA were used as templates for ORNi-PCR. In this regard, the EpiTect PCR Control DNA Set (QIAGEN, Valencia, CA, USA) includes CpG-methylated and bisulfite-treated DNA, and unmethylated and bisulfite-treated DNA. gDNA was extracted from the 293T and HCT116 cell lines and subjected (500 ng of each) to bisulfite treatment using the EZ DNA Methylation-DirectTM Kit (Zymo Research). Alternatively, 293T gDNA (500 ng) was mixed with HCT116 gDNA so that CpG-methylated CDKN2A (p16) accounted for 0–5% of the total CDKN2A (p16), and then the mixture was subjected to bisulfite treatment. After the measurement of its concentration, bisulfite-treated DNA was diluted and used for ORNi-PCR. ORNi-PCR was performed using KOD -Multi & Epi-TM (Toyobo, Osaka, Japan) in mixtures containing 100 fg plasmid DNA or 10 ng bisulfite-treated gDNA, 0.3 μM of each primer, and 1–6 μM ORN in a total volume of 10 μL. ORNi-PCR was performed at various temperatures for the annealing and elongation steps in the presence of different concentrations of ORNs along with a method to determine the optimal conditions [7]. An established optimal condition is shown in Figure 6D.

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