Abstract
DNA methylation is an important mode of epigenetic modification, which plays key roles in many cellular processes. Within the last decades, various methods have been proposed for quantitative analysis of methylated DNA. However, the reports relating to the monitoring of dynamic methylation process are rare. The challenges of tracking DNA methylation process mainly include the detection of minute change during the methylation process and the synchronously amplifying of target signals. Herein, we propose an electrochemical strategy for continuous monitoring of DNA methylation process over time based on long-range electron transfer. An electrochemical sensor is prepared by assembling single-strand DNA probes whose tops are labeled with 6-ferrocenylhexanethiol-modified gold nanoparticle. No obvious current response can be observed until the establishment of the long-range electron transfer pathway between 6-ferrocenylhexanethiol and electrode by hybridization of the complementary DNA. Once the DNA is methylated, a bromine group will be immediately colocated onto it in the presence of NaIO4/LiBr. This derivatization causes the decline of the charge density around the mC•G base pair, following with an obvious current reduction. Owing to the velocity of the bromine derivatization is faster than that of the methylation; the general signal can promptly reflect the methylation status of the DNA. By continuously measuring the current decrease ratio, the monitoring of the dynamic process of DNA methylation can be achieved. In this chapter, we describe in detail the protocols of this method, including the label of DNA probe with electrochemical tags, the construction of the long-range electron transfer pathway on electrochemical sensor, the operation of the DNA methylation and bromine derivatization, as well as the continuous measurements of the conversed signals. This method may be potential for the application in biological research, disease diagnostics, and other areas.
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References
Dahl C, Guldberg P (2003) DNA methylation analysis techniques. Biogerontology 4:233–250
Tucker KL (2001) Methylated cytosine and the brain: a new base for neuroscience. Neuron 30:649–652
Ehrlich M, Gama MA, Huang LH, Midgett RM, Kuo KC, McCune RA et al (1982) Amount and distribution of 5-methylcytosine in human DNA from different types of tissues or cells. Nucleic Acids Res 10:2709–2721
Cottrell SE (2004) Molecular diagnostic applications of DNA methylation technology. Clin Biochem 37:595–604
Gertz J, Varley KE, Reddy TE, Bowling KM, Pauli F, Parker SL et al (2011) Analysis of DNA methylation in a three-generation family reveals widespread genetic influence on epigenetic regulation. PLoS Genet 7:E1002228
Jones PA, Laird PW (1999) Cancer epigenetics comes of age. Nat Genet 21:163–167
Trasler JM (2006) Gamete imprinting: setting epigenetic patterns for the next generation. Reprod Fertil Dev 18:63–69
Esteller M, Corn PG, Baylin SB, Herman JG (2001) A gene hypermethylation profile of human cancer. Cancer Res 61:3225–3229
Laird PW (2003) The power and the promise of DNA methylation markers. Nat Rev Cancer 3:253–266
Zhang L, Xu YZ, Xiao XF, Chen J, Zhou XQ et al (2015) Development of techniques for DNA-methylation analysis. Trends Anal Chem 72:114–122
Gonzalgo ML, Jones PA (1997) Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res 25:2529–2531
Aggerholm A, Guldberg P, Hokland M, Hokland P (1999) Extensive intra and inter individual heterogeneity of pl5INK4B methylation in acute myeloid leukemia. Cancer Res 59:436–441
Fraga MF, Uriol E, Borja LD, Berdasco M, Esteller M, Cañal MJ et al (2002) High-performance capillary electrophoretic method for the quantification of 5-methyl 2-deoxycytidineingenomic DNA: application to plant, animal and human carcinoma tissues. Electrophoresis 23:1677–1681
Rodríguez-Lopez CM, Guzmán AB, Lloyd AJ, Wilkinson MJ (2010) Direct detection and quantification of methylation in nucleic acid sequences using high resolution melting analysis. Anal Chem 82:9100–9108
Zhang J, Song J, Nie C, Liu L, Lv F, Wang S et al (2014) Associated analysis of DNA methylation for cancer detection using CCP-based FRET technique. Anal Chem 86:346–350
Xu YZ, Niu C, Xiao XF, Zhu WY, Dai Z, Zou XY (2015) Chemical-oxidation cleavage riggered isothermal exponential amplification reaction for attomole genespecific methylation analysis. Anal Chem 87:2845–2851
Daems D, Knez K, Delport F, Spasica D, Lammertyn J (2016) Real-time PCR melting analysis with fiber optic SPR enables multiplex DNA identification of bacteria. Analyst 141:1906–1911
Huang W, Qi CB, Lv SW, Xie M, Feng YQ, Huang WH, Yuan BF (2016) Determination of DNA and RNA methylation in circulating tumor cells by mass spectrometry. Anal Chem 88:1378–1384
Meissner A, Gnirke A, Bell GW, Ramsahoye B, Lander ES, Jaenisch R (2005) Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res 33:5868–5877
Wang CH, Lai HC, Liou TM, Hsu KF, Chou CY, Lee GB (2013) A DNA methylation assay for detection of ovarian cancer cells using a HpaII/MspI digestion- based PCR assay in an integrated microfluidic system. Microfluid Nanofluid 15:575–585
Kelley OS, Barton KJ (1999) Electron transfer between bases in double helical DNA. Science 283:375–381
Lewis DF, Wu TF, Zhang YF, Letsinger LR, Greenfield RS, Wasielewski RM (1997) Distance-dependent electron transfer in DNA hairpins. Science 277:673–667
Mohamadi M, Mostafavi A, Torkzadeh-Mahani M (2015) Electrochemical determination of biophenol oleuropein using a simple label-free DNA biosensor. Bioelectrochemistry 101:52–57
Claesson MJ, Cusack S, O’Sullivan O, Greene-Diniz R, Weerd HD, Flannery E et al (2016) Highly specific SNP detection using 2D graphene electronics and DNA strand displacement. Proc Natl Acad Sci U S A 113:7088–7093
Bareyt S, Carell T (2008) Selective detection of 5-methylcytosine sites in DNA. Angew Chem Int Ed 47:181–184
Acknowledgments
This work was supported by the National Natural Science Foundations of China (21422510, 21375154, and 21675180), the Scientific Technology Project of Guangdong Province (2016B010108007, 2014A040401022, 2015A030401033, and 2017B020221001), and the Scientific Technology Project of Guangzhou City (201604020145).
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Dai, Z. et al. (2022). Electrochemical Assay for Continuous Monitoring of Dynamic DNA Methylation Process. In: Yuan, BF. (eds) DNA Modification Detection Methods . Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1229-3_6
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DOI: https://doi.org/10.1007/978-1-0716-1229-3_6
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