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Colorimetric and electrochemical quantification of global DNA methylation using a methyl cytosine-specific antibody

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Abstract

We report a simple colorimetric (naked-eye) and electrochemical method for the rapid, sensitive and specific quantification of global methylation levels using only 25 ng of input DNA. Our approach utilises a three-step strategy; (i) initial adsorption of the extracted, purified and denatured bisulfite-treated DNA on a screen-printed gold electrode (SPE-Au), (ii) immuno-recognition of methylated DNA using a horseradish peroxidase (HRP)-conjugated methylcytosine (HRP-5mC) antibody and (iii) subsequent colorimetric detection by the enzymatic oxidation of 3,3′,5,5′-tetramethylbenzidin (TMB)/H2O2 which generated a blue-coloured product in the presence of methylated DNA and HRP-5mC immunocomplex. As TMB(ox) is electroactive, it also produces detectable amperometric current at +150 mV versus a Ag pseudo-reference electrode (electrochemical detection). The assay could successfully differentiate 5-aza-2′-deoxycytidine drug-treated and untreated Jurkat DNA samples. It showed good reproducibility (relative standard deviation (% RSD) = <5%, for n = 3) with fairly good sensitivity (as low as 5% difference in methylation levels) and specificity while analysing various levels of global DNA methylation in synthetic samples and cell lines. The method has also been tested for analysing the methylation level in fresh tissue samples collected from eight patients with oesophageal squamous cell carcinoma. We believe that this assay could be potentially useful as a low-cost alternative for genome-wide DNA methylation analysis in point-of-care applications.

Graphical abstract: Colorimetric and electrochemical quantification of global DNA methylation using a methyl cytosine-specific antibody

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Publication details

The article was received on 28 Mar 2017, accepted on 25 Apr 2017 and first published on 26 Apr 2017


Article type: Paper
DOI: 10.1039/C7AN00526A
Citation: Analyst, 2017, Advance Article
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    Colorimetric and electrochemical quantification of global DNA methylation using a methyl cytosine-specific antibody

    Md. H. Haque, R. Bhattacharjee, Md. N. Islam, V. Gopalan, N. Nguyen, A. K. Lam and M. J. A. Shiddiky, Analyst, 2017, Advance Article , DOI: 10.1039/C7AN00526A

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