Electrochemical DNA methylation detection for enzymatically digested CpG oligonucleotides

We describe the electrochemical detection of DNA methylation through the direct oxidation of both 5-methylcytosine (mC) and cytosine (C) in 5'-CG-3' sequence (CpG) oligonucleotides using a sputtered nanocarbon film electrode after digesting a longer CpG oligonucleotide with endonuclease P1. Direct electrochemistry of the longer CpG oligonucleotides was insufficient for obtaining the oxidation currents of these bases because the CG rich sequence inhibited the direct oxidation of each base in the longer CpG oligonucleotides, owing to the conformational structure and its very low diffusion coefficient. To detect C methylation with better quantitativity and sensitivity in the relatively long CpG oligonucleotides, we successfully used an endonuclease P1 to digest the target CpG oligonucleotide and yield an identical mononucleotide 2'-deoxyribonucleoside 5'-monophosphate (5'-dNMP). Compared with results obtained without P1 treatment, we achieved 4.4 times higher sensitivity and a wider concentration range for mC detection with a resolution capable of detecting a subtle methylated cytosine difference in the CpG oligonucleotides (60mer).