Abstract
Aberrations of genomic DNA methylation have been confirmed to be involved in the evolution of human cancer and have thus gained the potential to be depicted as biomarkers for cancer diagnostics and prognostic predictions, which implicates an urgent need for detection of total genomic DNA methylation. In this work, we suggested an assay for the quantification of global DNA methylation, utilizing methylation specific antibody (5mC) modified magnetic beads (MBs) for immunorecognition and affinity enrichment. Subsequently, the captured DNA on the surface of MBs interacted with the glucose oxidase-conjugated DNA antibody whose catalytic reaction product was engaged in electrochemical detection of the overall level of DNA methylation on a PB-doped screen-printed electrode. With 15 pg of input DNA, which, to our best knowledge, is the lowest required amount of DNA without sodium bisulfite treatment or amplification, this test strategy was able to perceive as low as 5% methylation level within 70 min including the preparation of anti-5mC-MBs. We believe this detection technique offers a promising option to detect global DNA methylation in both academic and clinical scenarios.
Highlights
Introduction of Genomic DNA MethylationAs one of the major epigenetic modifications of DNA, DNA methylation, which is that a methyl group (-CH3 ) was added to the fifth carbon of a cytosine following a guanine nucleotide (CpG sites), is known to be crucial in gene expression regulation and involved in various cellular processes, including development and disease [1–4].Alteration of 5-methylcytosine (5mC) at global levels is more resistant than that in selected loci [5]
Before screen printed Prussian blue (PB) electrodes were applied to detect the product generated from oxidase catalyzed reactions, their catalytic activity towards H2 O2 was explored
The concentration range of H2 O2 reduction test conducted at the surface of PB electrode was chosen as 0–0.5 mM because the quantity of H2 O2 produced by glucose oxidase (GOx) was deemed to be small
Summary
Introduction of Genomic DNA MethylationAs one of the major epigenetic modifications of DNA, DNA methylation, which is that a methyl group (-CH3 ) was added to the fifth carbon of a cytosine following a guanine nucleotide (CpG sites), is known to be crucial in gene expression regulation and involved in various cellular processes, including development and disease [1–4].Alteration of 5-methylcytosine (5mC) at global levels is more resistant than that in selected loci [5]. As one of the major epigenetic modifications of DNA, DNA methylation, which is that a methyl group (-CH3 ) was added to the fifth carbon of a cytosine following a guanine nucleotide (CpG sites), is known to be crucial in gene expression regulation and involved in various cellular processes, including development and disease [1–4]. There are mainly three categories of strategies utilized to distinguish methylated from unmethylated DNA—sodium bisulfite conversion, methylation-sensitive restriction enzymes (MSREs) and affinity-based techniques. Sites that can be analyzed are highly confined by the cleavage site of available MSREs and they may not be able to identify other epigenetic modifications of DNA, such as 5hydroxymethylcytosine (5hmC) [15]. It is not required to have the knowledge of specific sequences in order to determine the global DNA methylation level
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