Figure 2.

Separation of PCR products amplified from the synthetic DNA fragments by the newly developed anion‐exchange high‐performance liquid chromatography method. A, Chromatograms of synthetic DNA fragments 1, 3, 5, 6, 7, 8, 9 and 10 corresponding to the sequences after bisulfite modification of 0%, 25% 50%, 75% and 100% methylated promoter regions of the FAM150A gene (Table 1). Flow rate, 1.0 mL/min; buffer A, 25 mmol/L Tris‐HCl, pH 7.5; buffer B, 1 mol/L NH ₄(SO ₄)₂ in buffer A; linear gradient from 40%‐100% buffer B in 10 minutes; column temperature, 70°C; injection volume, 5 μL. B, Enlarged view of the chromatograms of (A); retention time: 7‐8.4 minutes. C, Linearity of the decrease in the retention time associated with an increase in the methylation level from 0% to 100%. D, Within‐run reproducibility test using PCR products obtained from Fragments 2, 3 and 4 corresponding to the sequences after bisulfite modification of 20%, 25% and 30% methylated promoter regions of the FAM150A gene (Table 1). Error bar: SD