INTRODUCTION CpG methylation of the human genomic DNA plays an important role in gene regulation. Recently, it has been reported that aberrant CpG methylation is related to human diseases such as cancer. Therefore, these modifications at specific genes are regarded as new biomarkers. To detect methylation of DNA, usually, bisulfite sequencing is used. However, it is time consuming and laborious. Therefore, to target CpG methylation for diagnosis, a more simple and rapid detection system is required. Therefore, in this study, we tried to develop a new system for CpG methylation. METHODS To construct simple system, we focused on Methyl CpG-binding domain (MBD), and glucose dehydrogenase (GDH) fused zinc finger protein (ZFP) (Fig.). As MBD can bind to methylated double strand DNA (dsDNA), we can separate methylated DNA without denaturing process. Then, after separation and PCR amplification, we detected the PCR products by zinc finger protein. As ZFP can bind to its target dsDNA, we can detect specific target sequence by just observing specific binding of ZFP. In terms of diagnosis, electrochemical detection systems have several advantages including a rapid response and miniaturization. Therefore, we chose glucose dehydrogenase as a label for ZFP. We tried to check if this system works. As a model target region, we chose androgen receptor (AR). Abnormal epigenetic modifications in AR region is regarded as a diagnostic index for prostate cancer. To detect CpG methylation level at AR region, First, we made calibration graph using 106 copies of genomic DNA with different methylation level. Next, using the graph, we determined the CpG methylation level in PC3 and TSU-PR1 cancer cells. RESULT and DISCUSSION When we made calibration, the current response was increased dependent on the methylation level even the amount of genomic DNA is same among the evaluated genomic DNA, indicating our system is responding to methylation level. Then, using the calibration we detected the CpG methylation level at AR region. The detected methylation levels showed high correlation with the results obtained by conventional method. That means CpG methylation level was correctly detected without laborious, time-consuming conventional method in just 3 h.

Development of an electrochemical detection system for CpG methylation of human genome using methyl CpG binding domain and zinc finger protein

256th National Meeting (2018)

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