Methylation Detection Using MALDI-TOF MS

MALDI-TOF MS spectra for the detection of different DNA methylation levels (examples shown: 100% methylated DNA; 50:50 mix of methylated and unmethylated DNA; 10% methylated DNA samples mixed with 90% unmethylated DNA) after bisulfite treatment in specific CpG positions in the promoter of INK4A (p16) gene (specific amplicon mapping to Chromosome 9: 21964960–21965128 on the Human Genome May 2004 assembly).

(A) Primer extension approach using iPLEX chemistry to detect DNA methylation at CpG 2 (Chromosome 9: 21965105–21965106), C call corresponding to the methylated and T call to the nonmethylated alleles.

(B) MassCLEAVE assay using T-reverse specific cleavage, detection of the methylation levels at CpG 10–11 (Chromosome 9: 21964989–21964992 on the Human Genome May 2004 assembly). Fragments detected on the reverse strand are as follows: 4484.85 Dalton 5OH-AAAAAAACCACAAT-3p for the unmethylated (Unmeth) and 4516.84 Dalton 5OH-AAAAAAACCGCGAT-3p with both CpG sites methylated (Meth).