| Epigenetic processes, which are concerned with inherited changes in gene expression or cellular phenotype that do not rely on alteration to the underlying DNA sequence,are associated with the regulation of gene expression through the chemical tagging of chromatin and DNA,which can alter their interaction with transcription factors. Historically, the most widely studied modification of DNA is the 5-methycytosine, which is catalyzed by DNA methyltransferases(DNMTs) and occurs at cytosine-5-position(5-mC), usually within CpG dinucleotides. DNA methylation is essential in regulating gene expression in nearly all biological processes including development, growth, and differentiation. DNA methylation is widely studied in the context of cancer. However, the discovery of 5-hydroxymethylation of DNA adds new layer of complexity to understanding the epigenetic basis of the development and disease, including carcinogenesis. There has been a grow body of inquiry into the function, dysregulation and potential significance of 5-hydroxymethycytosine(5-hmC) in physiology and pathology. Therefore, it is crucial to develop novel sensitive and selective methods for detection of DNA methylation and hydroxymethylation.Electrochemical biosensors are widely studied and applied in cancer related biomarker prognostic, medical health screening, environmental quality monitoring, and some other related areas due to its rapid speed,high sensitivity, and low cost. This paper focused on nano-probe based electrochemical sensor design and developed several sensitive 5-mC and 5-hmC detection electrochemical methods, as well as applying in original sample detection. 1 DNA nanostructure-based electrochemical methylation-specific PCR biosensor(E-MSP) for methylation detectionTetrahedral DNA nanostructure has a much thicker layer, and places the probes in a solution-phase-like environment with enhance capture-target probe binding affinity. E-MSP combines the high specificity of MSP with high sensitivity and simplicity of 3D DNA nanostructure-based electrochemical technology. E-MSP facilitates a straightforward method for quantitative detection of methylation DNA.For the first time, the sensitivity of the E-MSP is examined in simulated samples(methylated plasmid). Notably, electrochemical signals were observed using as little as 160 fg of methylation DNA in an excess of 160 ng of unmethylation DNA, which is distinctly above the signal observed with only unmethylation DNA. Most importantly, signal observed from E-MSP from the 1:1000000 dilution samples indicates a clear difference from the unmethylation control. 2 Noninvasive detection and differential diagnosis of prostate cancer using associated GSTP1 and TNFR10 D genes by E-MSP measurement of promoter methylationThe unique 3D DNA nanostructure-based electrochemical MSP system provides high detection sensitivity, excellent specificity and low background noise. As reported, Cell-free DNA from the tumor or affected tissues represent a small minority of the sample, with most DNA coming from reactive inflammatory cells and healthy cells. The high sensitivity of E-MSP strategy have been proved with the detection of only 0.0001% of original methylation gene for remarkable signal to noise. More importantly, we have demonstrated a cumulative detection mode can be used to accurate and sensitive distinguish prostate cancer and BPH which will be essential to perform anti-cancer therapy and assess prognosis. We analyzed quantitatively the methylated DNA levels of cell-free DNA in serum from patients with prostate cancer, BPH and normal control in Chinese population. Through discriminate analysis and combination mode, a high sensitivity of 94.1% was obtained for patients with cancer and normal control, 66.7% for BPH cases and normal control. 3 High-efficiency Detection of DNA Hydroxymethylation in Genomic DNA by Multiplexing Electrochemical BiosensingA sensitively and selectively MCE biosensor was developed for 5-hmC detection in total genomic DNA based on chemical modification of hydroxyl in 5-hm C triggered enzymatic signal amplification. The developed method presented high detection specificity because the chemical reaction for 5-hmC modification can not happen at other unhydroxymethylated nucleic acids bases. As a proof of concept investigation, this method also showed high detection sensitive with low LOD of 0.489pg(0.0012%) for 5-hmC in total DNA. Our quantification results in different tissues show that 5-hmC is present in every tissue investigated. The highest level of 5-hmC is detected in DNA isolated from brain(0.163%). DNA extracted from kidney, heart, and lung has medium 5-hmC values from 0.045% to 0.06%. DNA from liver and spleen possess the lowest amount of 5-hmC. As expected, DNA from all cell lines possessed very low amount of 5-hmC, which is agreed with most reports. 4 Multiplexing Electrochemical sensor for hydroxymethylation detection at specific gene lociThe role of hydroxymethylcytosine in gene regulation is draw much attention. Notably, a line of evidence showed that conversion of 5-mC to 5-hmC greatly reduced affinity of MBD proteins to methylated DNA, and 5-hmC accounts for roughly 40 percent of the methylated cytosine in purkinje cells and is also specifically localized in CpG region. Because of the presence of 5-hmC in DNA with unclear function and the discovery of the TET enzymes that produce 5-hmC, it is considered necessary to determine the location of this modification in different gene region. So we used KRuO4 to oxide hydroxymethycytosine, which could be oxided as aldehydemethycytosine. Followed, sodium bisulfite was used to treat the same sequence of DNA, resulting aldehydemethycytosine transferred as thymine(T). Finally, the product could exist mismatch at the position of hydroxymethycytosine. Importantly, electrochemical sensor can detect this mismatch by T7 endonuclease 1 cutting the product. This project can be used to analyze 5-hmC changes during cell differentiation, physiological and pathological process. |
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