基于内切酶特异性剪切作用的疾病DNA电化学分析方法研究

【作者】 刘姝娜；

【导师】 蔡称心；

【作者基本信息】 南京师范大学 ， 生物物理化学， 2011， 博士

【摘要】 随着国民经济和社会的不断发展、生活水平的不断提高,人们对自身健康的要求也越来越高,但各种疾病仍是危害人类健康的重要因素之一,它的发病和死亡率上升己引起人们的普遍关注,对各种疾病进行早期诊断和治疗对于提高疾病患者的生存率具有十分重要的意义。因此,早期诊断和疗效观察方法的研究已成为疾病防治研究领域的重要前沿课题之一。本论文旨在建立某些疾病DNA的电化学检测新方法,为某些疾病的临床诊断和疗效观察提供快速、准确和灵敏的方法。主要研究思路是将设计的探针DNA固定在适合的电极表面,然后将特定的电化学指示剂通过共价键合或特性吸附作用固定到探针DNA上；使探针DNA与具有特定疾病基因特征的DNA片段杂交,再经特异性内切酶作用后,记录指示剂分子电化学信号的变化,从而检测疾病DNA。本论文工作主要以丙型肝炎病毒(HCV)和DNA甲基化引起的恶性疾病基因为研究对象,建立HCV的电化学检测方法,实现DNA甲基化检测、DNA甲基化酶活性分析以及与DNA甲基化相关抗癌药物的筛选。主要内容如下：1.利用限制性内切酶对特定DNA序列的特异性识别和剪切作用,建立了一种HCV特异性分析和检测的电化学方法。该方法是将电化学指示分子硫堇(Thionine)标记的特定序列DNA探针固定在Au电极表面,与目标cDNA(该目标cDNA与HCV的DNA序列有关)进行杂交,然后经限制性内切酶BamHI剪切。剪切后,电化学指示分子硫堇的电化学响应电流减小或消失,电流减小的幅度(Δi)与杂交的目标cDNA浓度有关,因而可实现HCV的电化学检测与分析。我们对各种可能影响检测的因素进行了详细研究,结果表明,在最佳条件下,Δi与目标cDNA在0.1-2.5μmol/L浓度范围内呈线性关系,最低检测限为0.02μmol/L(S/N=3)。并且,该方法能灵敏地分辨出完全互补和只有一个碱基错配的目标cDNA,具有好的选择性,并且能实现实时监测。2.在前面的研究基础上,选用氧化还原可逆特性较好的羧基二茂铁(FcA)作为电化学指示分子,研究了所建立方法对HCV的电化学检测作用,目的是进一步提高HCV的检测灵敏度,降低其检测限。该方法是先将特定序列的探针DNA固定在Au电极表面并经电化学指示分子FcA标记,再与目标cDNA(该目标cDNA与HCV的DNA序列有关)进行杂交,然后被限制性内切酶BamHI剪切,根据氧化还原指示分子FcA的微分脉冲伏安(DPV)峰电流的变化(Δi)进行检测。用循环伏安及交流阻抗方法对探针DNA在Au电极表面的固定过程进行了表征,用循环伏安及DPV方法对FcA与探针DNA的作用进行了研究,并且,详细研究了杂交时间、离子强度、温度、溶液pH对探针DNA与目标cDNA杂交的影响,实验结果表明,Δi值与HCV DNA(cDNA)在0.05-4.0μmol/L范围内呈线性关系,最低检测限为(0.5±0.2)nmol/L(S/N=3)。该方法能用于识别只有1个错配碱基的目标cDNA并可用于实际样品中HCV的定量检测,具有简单、灵敏度高、特异性好、可实时监测等优点,理论上,只要简单地改变探针DNA的序列及限制性内切酶的种类,该方法就能用于分析检测其他类型的基因型病毒,从而发展成一种通用的DNA分析方法。3.HCV基因分型对不同基因亚型病毒特异疫苗的研制及临床治疗药物的筛选等都有着十分重要的意义。我们发展了一种基于限制性内切酶BamHI联合金纳米颗粒(AuNPs)信号放大的HCV型别的电化学检测新方法。该方法是将硫堇-探针DNA固定到电极表面,并与HCV 1b型目标cDNA (244-mer)杂交,再经限制性内切酶BamHI剪切,记录剪切前后硫堇DPV峰电流的变化。结果表明,DPV峰电流(Δi)与目标cDNA在1×10-21-1×10-10mol/L浓度范围内呈线性关系,最低检测限达(3.1±0.8)×10-22 mol/L(S/N=3),与常用的罗氏检测HCV试剂盒方法相比(最低检测限是2.7×10-18mmol/L),检测限低4个数量级。而且,该方法还能用于从HCV 1、HCV1a HCV 1b、HCV 6a型中选择性地分辨出HCV 1b型,因而可用于HCV基因型别的检测。4.发展了一种能用于DNA甲基化的检测、DNA甲基化转移酶活性的分析以及与DNA甲基化相关抗癌药物筛选的电化学方法。该方法是将FcA与探针DNA的3,端共价键合,与目标cDNA杂交后经甲基转移酶(M. SssI)将CpG序列中的胞嘧啶(C)甲基化,并经限制性内切酶HpaII剪切,未甲基化的DNA在特定位点被剪切,而甲基化的DNA不被剪切,根据FcA电化学信号在剪切前后的变化检测DNA甲基化水平、分析甲基化酶活性。我们还研究了抗癌药物5-氮杂胞嘧啶核苷(5-Aza)和5-氮杂-2’-脱氧胞嘧啶核苷(5-Aza-dC)对甲基转移酶活性的抑制作用,表明该方法可用于评估抗癌药物对DNA甲基化酶的抑制作用,并且可用于相关抗癌药物的筛选。更多还原

【Abstract】 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.更多还原