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The levels of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in DNA (5-mC-DNA and 5-hmC-DNA) are strongly correlated with cancer occurrence and development. The ability to distinguish and quantitatively detect them is important for cancer research. We have developed a hybridization chain reaction (HCR)-based electrochemical assay for the signal-amplified detection of the relative contents of 5-mC-DNA and 5-hmC-DNA. The DNA duplexes (containing 5-mC-DNA and 5-hmC-DNA with different percentages) were modified on a gold electrode. Electroactive [Ru(NH3)6]3+ (RuHex) was used as the signal reporter, because it binds to DNA double strands. The duplexes can be cleaved by MspJI endonuclease without HCR, and result in a small peak current. However, the cleavage can be blocked after the 5-hmC-DNA duplex is converted to β-glucosyl-5-hydroxymethylcytosine (β-glu-5-hmC) by T4 β-glucosyltransferase (T4 β-GT), and with the addition of helper DNA, a long double-helix DNA was formed through HCR. A significantly amplified peak current can be achieved due to the adsorption of numerous RuHex. The electrochemical signal of RuHex is correlated to the content of 5-hmC-DNA. Upon fixing the total quantity of 5-mC-DNA and 5-hmC-DNA on the electrode, the signals increase with the increase in the percentage of 5-hmC-DNA for the HCR. With this assay, a detection limit of 0.05% for 5-hmC-DNA was achieved.

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