MR-SNuPE assay design for SEPT9 and general performance. (A) SEPT9 amplicon sequence with indicated primer (boxes) and blocker (line above) positions. (B) Electropherograms of separated single nucleotide primer extension (SNuPE) products from PCR products obtained without blocker on (a) completely methylated and (b) unmethylated DNA templates or (c-f) mixed DNA templates (methylated:unmethylated DNA ratios/dilution series). (g) Effect when performing a heavy methyl (HM)-PCR; that is, when the blocker is included, on a dilution shown in (f). Vertical dashed lines indicate the positions of unextended primer, methylated and unmethylated signals, respectively, NTC = no template control, that is, SNuPE reaction without PCR template.