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Electrochemical immunoassays for the detection the activity of DNA methyltransferase by using the rolling circle amplification technique

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Abstract

We report on an electrochemical method for the determination of the activity of the enzyme methyltransferase (MTase). The methyl-binding domain-1 protein was applied to recognize symmetrically methylated cytosine in CpG (-C-phosphate-G-) islands of ds-DNA which then specifically bind to anti-His tag antibody. Hyperbranched rolling circle amplification (RCA) was used to improve sensitivity. When the dsDNA was treated with M.Sss I methyltransferase, the sequence 5′-CCGG-3′ was methylated and recognized by the methyl binding protein. In turn, the anti-His tag, biotinylated IgG, streptavidin and biotinylated oligonucleotide were captured successively on the surface of an electrode. Subsequently, the RCA reaction was initiated and streptavidin-labeled alkaline phosphatase immobilized on the surface of the electrode. ALP was able to catalyze the hydrolysis of 1-naphthyl phosphate to form 1-naphthol at pH 9.8. The oxidation peak current of 1-naphthol was used to monitor the methylation process. The response obtained by differential pulse voltammetry was linearly related to the concentration of M.Sss I MTase in the range from 0.1 to 40 unit mL−1, and the detection limit was 0.03 unit mL−1 (at an SNR of 3). The inhibitory action of paclitaxel on the activity of M.Sss I MTase also was investigated.

An electrochemical immunosensing method is described for the detection of DNA methylation, assaying DNA methyltransferase activity by combining the rolling circle amplification technique.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 21075078, 21105056) and the Natural Science Foundation of Shandong province, China (No. ZR2010BM005, ZR2011BQ001).

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Authors and Affiliations

  1. College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong, 271018, People’s Republic of China

    Zhenning Xu, Huanshun Yin, Zhengbin Tian, Yunlei Zhou & Shiyun Ai

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  1. Zhenning Xu
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  2. Huanshun Yin
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  3. Zhengbin Tian
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  4. Yunlei Zhou
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  5. Shiyun Ai
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Correspondence to Yunlei Zhou or Shiyun Ai.

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Xu, Z., Yin, H., Tian, Z. et al. Electrochemical immunoassays for the detection the activity of DNA methyltransferase by using the rolling circle amplification technique. Microchim Acta 181, 471–477 (2014). https://doi.org/10.1007/s00604-013-1141-1

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  • DOI: https://doi.org/10.1007/s00604-013-1141-1

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