Dna Electrochemical Analysis Methods Based On The Enzyme Specificity For Shear Stress In Disease Research

The aim of this thesis is to develop a electrochemical approach used for detection of hepatitis C virus (HCV), the determination of DNA methylation level, analysis the activity of methyltransferase, and evaluation and screen of the inhibitors of methyltransferase. As a general method of DNA detection, these studies are expected to be applicable to other type of DNA analysis. It has high potential application in molecular diagnostics of disease in clinical environments, and may be helpful for the discovery of anticancer drugs. The main results are as follows:1. An electrochemical method for detection of oligonucleotides related to HCV sequence based on the site-specific cleavage by BamHI endonuclease is described. Thionine covalently linked to the probe DNA sequence is used as indicator. The thionine-labeled probe DNA was self-assembled on the surface of gold electrode and hybridized with target cDNA (an oligonucleotide related to HCV), then digestion with BamHI endonuclease. Electrochemical assay is performed by monitoring the alteration (△i) of the voltammetric signal of thionine before and after the hybridized DNA is digested by BamHI. Under the optimum condition, the value of△i has a linear relationship with the concentration of target cDNA in the region of 0.1 to 2.5μmol/L with a detection limit of 0.02μmol/L (at S/N=3). The proposed model displays a good selectivity by discriminating one-base mismatched cDNA sequence, and the ability to perform real-time monitoring.2. Our previous study reported a preliminary study on the detection of HCV based on site-specific DNA cleavage of BamHI endonuclease using thionine-labeled probe. In this chapter, the further characterization and optimization of this new electrochemical sensing approach was continued with ferroceneacetic acid (FcA) as an electroactive label. FcA was selected as a label in this work since ferrocene and its derivatives have been considered as excellent probes in electrochemical DNA assays owing to their redox reversibility and synthetic versatility, the ability to promote the detection performance, and so forth. The method was developed by immobilizing a synthetic probe DNA on the surface of gold electrode via the -SH group at the 5'-terminus of the probe, and conjugating the electroactive label of FcA moiety to the 3'-terminus of the probe via formation of covalent bond between the -NH2 and -COOH groups. The FcA-labeled probe was then hybridized with target cDNA (an oligonucleotide related to HCV) and cleaved by BamHI endonuclease. It was demonstrated that the value of△i has a linear relationship with the concentration of the HCV DNA (cDNA) ranging from 0.05 to 4.0μmol/L, and the detection limit of (0.5±0.2) nmol/L at a signal/noise of 3. Moreover, it was successfully used in distinguishing the complementary sequence from the one-mismatched sequences with stability and reproducibility, and can also be used for detection of HCV in real clinical samples. The developed electrochemical approach, which is based on site-specific DNA cleavage of BamHI endonuclease, not only can qualitative and quantitative detect HCV, but also exhibits the advantages of ease of performance, good specificity and selectivity, and the ability to perform real-time monitoring. The developed protocol can be taken as a general method of DNA detection and is expected to be applicable to other type DNA analysis.3. This chapter proposes a new strategy for the electrochemical detection of HCV RNA level and identification of HCV 1b genotype based on the site-specific cleavage of BamHI endonuclease combined with gold nanoparticles (AuNPs) signal amplification. A 244-mer cDNA from HCV 1a, HCV 1b, HCV 1, and HCV 6a, respectively, was analyzed using a synthetic 21-mer DNA probe, which has been assembled on the electrode surface via a bifunctional molecule of p-aminobenzoic acid (ABA). The results demonstrated that the developed approach can be used for specifically identification of the HCV 1b genotype and selective and sensitive detection of HCV 1b cDNA (244-mer) with a detection limit as low as (3.1±0.8)×10-22 mol/L (less than 200 molecules). Moreover, the developed method has an ability to discriminate the HCV 1b cDNA sequence from even single-base mismatched DNA sequence, to assay the HCV 1b cDNA level precisely from the mixture of HCV 1, HCV 1b, HCV 1a, and HCV 6a, and to detect HCV in real clinical samples. The protocol has high potential application in molecular diagnostics of HCV in clinical environments.4. This chapter reports an electrochemical approach for the assay of MTase activity and the detection of DNA methylation of specific CpG sites. This approach is based on the voltammetric response of electroactive label (ferroceneacetic acid, FcA), which was conjugated to the 3'-terminus of the DNA, after the DNA hybrid was methylated by methyltransferase (M. Sssl) and further cleaved by HpaII endonuclease. After digested by Hpall, DNA hybrid was cleaved at specific site and electrochemical signals of FcA were decreased or disappeared. However, the cleavage of the endonuclease is blocked by CpG methylation. Therefore, the voltammetric signal after cleavage is related to the methylation status and MTase activity, which forms the basis of the assay of MTase activity and the determination of DNA methylation. The calibration curve indicates that the linear range of M. SssI assay is from 0.5 to 355 U/mL. The detection range of the developed method is much wider than that of previously reported MTase assays based on colorimetric approaches. The detection limit is estimated to be (0.1±0.02) U/mL at a signal/noise of 3. The validity of our developed method in evaluating and screening the inhibitors of M. Sssl was demonstrated by using 5-azacytidine (5-Aza) and 5-aza-2'-deoxycytidine (5-Aza-dC) as model inhibitors. In addition, This work develops an electrochemical approach for rapid detection of genomic DNA methylation level, assay of methyltransferase activity, and evaluation and screen of the inhibitors of methyltransferase. The screening of the inhibitors of MTase can be achieved based on the developed method and may be help for the discovery of anticancer drugs.