title: Electrochemical DNA methylation detection for enzymatically digested CpG oligonucleotides.
abstract: 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).
authors: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors: Dai
authors: D
authors: Kato
authors: Keisuke
authors: K
authors: Goto
authors: Shin-ichiro
authors: S
authors: Fujii
authors: Akiko
authors: A
authors: Takatsu
authors: Shigeru
authors: S
authors: Hirono
authors: Osamu
authors: O
authors: Niwa
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis; D003596:Cytosine / Q000032:analysis; D019175:DNA Methylation; D055664:Electrochemical Techniques; D005656:Fungal Proteins / Q000378:metabolism*; D037742:Nanotubes, Carbon / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*
chemical_list: C408982:CPG-oligonucleotide; D005656:Fungal Proteins; D037742:Nanotubes, Carbon; D009838:Oligodeoxyribonucleotides; D044503:5-Methylcytosine; D003596:Cytosine; C526100:Nuclease P1, Penicillium citrinum; D015719:Single-Strand Specific DNA and RNA Endonucleases