DNA胞嘧啶的甲基化与去甲基化进展

DNA胞嘧啶甲基化调控基因的表达以及多种生物功能,如细胞分化。概念上,DNA去甲基化是将已甲基化DNA核苷转化为未修饰核苷,是DNA甲基化的逆向过程。但在生物体内,这是一个涉及多步反应的非常复杂的过程。本综述简要介绍了动植物中DNA胞嘧啶的甲基化与去甲基化的研究现状,并讨论了恶性肿瘤(癌症)、阿尔茨海默氏病、心血管疾病、肺纤维病变和进食障碍等多种疾病中的甲基化与去甲基化异常。     

基于甲基化核酸内切酶的甲基转移酶活性的电化学检测研究

由DNA甲基转移酶(DNMT1)催化的DNA甲基化在许多生物过程中起重要作用,包括基因转录,基因组印记和细胞分化~([1])。本文利用特异性酶切法成功开发了一种有效的测定DNMT1的电化学方法。首先,将硫醇修饰的双链DNA(dsDNA)自组装固定在金电极上,随后DNMT1识别dsDNA半甲基化序列并实现完全甲基化,最后通过甲基化特异性限制酶(BSSHⅡ)剪切,未发生完全甲基化的碱基序列将被剪切并移除电极表面。利用差分脉冲伏安法(DPV)分别测定剪切前后的亚甲基蓝(MB)还原电流。在最佳优化条件下,该方法线性范围为0.5 nM～50 nM,检测限为0.5 nM。这种方法可以简单有效地检测DNA甲基化和DNMT1活性,在DNA甲基化快速检测和基因毒理分析方面具有一定的实际意义。     

DNA甲基化电化学分析

DNA甲基化是目前研究最多的DNA表观遗传修饰之一.大量研究表明,DNA甲基化会引起DNA结构、稳定性以及DNA与蛋白质相互作用方式的改变,从而影响基因表达,进而引起多种神经退行性疾病、免疫系统疾病甚至癌症.因此,发展简易、灵敏、准确、可靠的方法进行DNA甲基化的分析是至关重要的.本文简单介绍了DNA甲基化的分析方法,重点综述了DNA甲基化的电化学分析方法,并对DNA甲基化的研究前景进行了展望.     

污染水产去甲基化表观遗传毒性EGFP评价方法

pEGFP-C3质粒经过体外人工甲基化处理后,被转染进入HepG2细胞以构建重组细胞株．以5-AZA为阳性去甲基化毒物与重组细胞共培养,通过亚硫酸氢钠测序法定量检测EGFP基因启动子区甲基化状态,通过实时定量PCR检测EGFP基因表达,借助流式细胞术和荧光摄片定量检测共培养细胞的绿色荧光强度,在DNA甲基化、EGFP基因mRNA表达、GFP蛋白等多个层次研究5-AZA染毒处理与其去甲基化能力和荧光表达改变的响应关系．对天津污染水产的去甲基化能力进行了实际样品测试．结果表明,5-AZA与重组细胞的DNA甲基化、基因表达、蛋白产物变化之间存在显著关联,具有较低的检出浓度和良好的重复性．天津污染海域的水产去甲基化能力较强．本文初步建立了一种污染物去甲基化表观遗传毒性评价方法．     

面向DNA甲基化修饰分析的质谱技术

DNA甲基化作为表观遗传修饰中一种重要的调控方式,通过调控基因的表达,从而影响机体内一系列的生物学过程。色谱-质谱法是研究DNA甲基化修饰的重要研究手段。随着对哺乳动物DNA甲基化的生物学功能的深入研究,应用于研究表观遗传修饰的手段与仪器设备越来越先进。为了对DNA修饰进行定性与定量的分析检测,除了高效液相色谱整合不同种类质量分析器的质谱联用（HPLC-MS）技术外,目前还开发应用了基质辅助激光解析质谱技术（MALDI-ToF-MS）和气相色谱-质谱联用技术（GC-MS）,从而极大拓展了DNA甲基化修饰研究的手段。本文对分析表观遗传DNA甲基化修饰的质谱技术发展进行综述,希望为DNA甲基化修饰分析提供有价值的研究策略。     

核酸甲基化检测方法进展

核酸甲基化是表观遗传学领域最重要的现象之一,对于基因的活化、基因印迹、染色体稳定性等有重要影响.在全基因组范围内和特定基因片段中,甲基化水平均应维持在各自的正常范围内,任何的甲基化水平失常都将导致相应生理功能的失调.由于核酸甲基化在生命体系里的重要性,基于全基因和特定基因的甲基化检测在学术界引起了广泛关注.目前已有多种生物学和化学的方法用于核酸甲基化检测.这里,我们重点总结近年来新发展的基于化学修饰和分析化学的核酸甲基化检测方法.     

基于高效液相色谱的喹啉类药物对刺参DNA甲基化水平的影响研究

建立了高效液相色谱/紫外法测定刺参组织全基因组DNA甲基化水平的方法,并运用此方法对喹噁啉药物处理过的刺参样品DNA甲基化水平进行分析。色谱条件为:色谱柱为ZORBAX SB-Aq(4.6 mm×250mm;5μm);柱温为30℃;检测波长为280 nm;进样量20μL;流动相为甲醇-7 mmol/L乙酸铵(7∶93),流速为1.0 mL/min。5-甲基脱氧胞苷和脱氧胞苷的质量浓度在0.05~1.0 mg/L范围内时,线性关系良好,相关系数(r)均为0.999 9。5-甲基脱氧胞苷和脱氧胞苷的检出限均为0.05 mg/L,在加标浓度为0.05,0.25,1.0 mg/L时,回收率为90.4%~100.6%,批内、批间相对标准偏差均小于4%。采用CTAB法提取刺参组织DNA,酶解后进行HPLC测定,结果表明:喹噁啉药物处理过的组织样品DNA甲基化水平低于对照组,该类药物通过改变DNA甲基化水平而影响基因的正常表达,可能是其产生遗传毒性的一种机制,建立的方法可以很好地用于全基因组DNA总甲基化水平的检测。     

液相色谱-串联质谱法测定生物样本全基因组DNA甲基化

建立了基于液相色谱-电喷雾串联质谱的分析方法,对生物样本中全基因组DNA甲基化水平进行定量测定.首先将DNA从生物样本中提取出来,将DNA片段酶解为单核苷,利用液相色谱-串联质谱测定胞嘧啶核苷和5-甲基胞嘧啶核苷的含量,从而计算出其全基因组DNA甲基化率.利用该法研究了暴露于全氟辛烷磺酸的L-02细胞、10例原发性肝癌病例血浆样本和10例对照血浆样本的全基因组DNA甲基化水平,得出了它们的总甲基化率变化的初步结果.本方法操作简单,具有很高的灵敏度和稳定性,为研究生物样本,尤其是临床上易得但DNA含量极低的血浆样本的总甲基化水平提供了思路.     

基于DNA四面体纳米结构探针的电化学生物传感器检测DNA甲基化

该文以DNA四面体纳米结构探针(TSP)为捕获探针,将辣根过氧化物酶标记的IgG抗体结合在纳米金颗粒表面(AuNPs-IgG-HRP)作为信号分子,构建了一种新型DNA甲基化电化学传感器。利用一步热变性法组装成TSP后,通过Au—S键固定在修饰纳米金颗粒的金电极表面,经过靶标DNA杂交、5-甲基胞嘧啶(5-mc)抗体及AuNPs-IgG-HRP结合后,用差分脉冲伏安法(DPV)进行检测。采用循环伏安法(CV)和电化学阻抗谱(EIS)对修饰电极的构建过程进行电化学表征。探究了杂交时间、5-mc抗体浓度、IgG-HRP加入体积、氢醌(HQ)和过氧化氢(H2O2)浓度对传感器的影响。在最佳条件下,该传感器对甲基化DNA的线性响应范围为1.0×10-15~1.0×10-10 mol/L,检出限(S/N=3)为4.4×10-16 mol/L。该传感器具有良好的选择性和稳定性,为DNA甲基化检测提供了新方法。     

甲基化作用增强了盐效应对M.L.DNA大分子构象的影响

本文用Hha I DNA甲基化酶为工具酶,制备了甲基化水平为2.39％的Micrococcus luteus DNA(天然的M.L.DNA是完全未甲基化的)。又以CD光谱、UV光谱及微量热法等手段,比较了甲基化和未甲基化的M.L.DNA在不同MgCl_2浓度下的[θ]值、A值及释热值的变化。发现随着MgCl_2浓度的增加,两种M.L.DNA的[θ]_(275)值均减少;而—[θ]_(220-250)值加大;A_(260)值则呈明显的减色效应;释热值却相应增加。这都说明MgCl_2的作用使M.L.DNA大分子在空间上趋于缩拢,且这个过程均持续约10min。同时发现:甲基化水平为2.39％的M.L.DNA对MgCl_2效应的敏感性要比末甲基化的M.L.DNA高50—70倍。     

DNA microarray: a high throughput approach for methylation detection

We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.     

Detection of DNA methylation by hyperbranched rolling circle amplification and DNA microarray

Aberrant DNA methylation of CpG sites has been confirmed to be closely associated with carcinogenesis.Based on the hyperbranched rolling circle amplification(HRCA) and microarray techniques,a new method for qualitative detection of methylation was developed.In the present study,padlock probes hybridize the sample DNA at the methylation site to form a probe-DNA complex which is ligated and digested simultaneously by methylation specific enzymes.Only at the methylated CpG site is the padlock probe ligated successfully to form a circle template for the HRCA reaction.Utilizing the method of 3-dimensional polyacrylamide gel-based microarray,the HRCA product will be immobilized on the slide to form a DNA microarray,which can universally hybridize the Cy3-labeled oligonucleotide probe to detect the methylation status of CpG sites.To control the false positive signals,DNA ligase and temperature of ligation/digestion are optimized.Methylation status of four CpG sites located in P15,Ecadherin,hMLH1 and MGMT genes were analyzed successfully with this method and all the results were compatible with that of methylation-specific PCR.Our research proves that this method is simple and inexpensive,and could be applied as a high-throughput tool to qualitatively determine the methylation status of CpG sites.     

Ferrocenylnaphthalene diimide-based electrochemical detection of methylated gene

Ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay was applied to the detection of methylated cytosine of DNA using the products obtained after treatment with bisulfite followed by polymerase chain reaction (PCR), where unmethylated cytosine is converted to thymine and methylated one to cytosine. Twenty-meric DNA probes for the methylated (cytosine) and unmethylated (thymine) types of the part of the promoter region of cyclin D-dependent protein kinase inhibitor, p16, gene (p16^Ink4a) were used to be immobilized on the electrochemical array (ECA) chip. Using 1 μL of 10 ng/μL of methylated sample obtained from the methylation-specific PCR of methylated genome containing 10-times excess of unmethylated one, the methylated PCR sample could be detected by the identical electrochemical signals from the two DNA probes under the settled optimum hybridization conditions.     

Allele-specific extension on microarray for DNA methylation analysis

Aberrant DNA methylation of CpG site in the gene promoter region has been confirmed to be closely associated with carcinogenesis. In this present study, a new method based on the allele-specific extension on microarray technique for detecting changes of DNA methylation in cancer was developed. The target gene regions were amplified from the bisulfite treated genomic DNA (gDNA) with modified primers and treated with exonuclease to generate single-strand targets. Allele-specific extension of the immobilized primers took place along a stretch of target sequence with the presence of DNA polymerase and Cy5-labeled dGTP. To control the false positive signals, the hybridization condition, DNA polymerase, extension time and primers design were optimized. Two breast tumor-related genes (P16 and E-cadherin) were analyzed with this present method successfully and all the results were compatible with that of traditional methylation-specific PCR. The experiments results demonstrated that this DNA microarray-based method could be applied as a high throughput tool for methylation status analysis of the cancer-related genes, which could be widely used in cancer diagnosis or the detection of recurrence.     

An osmium-DNA interstrand complex: application to facile DNA methylation analysis

Nucleic acids often acquire new functions by forming a variety of complexes with metal ions. Osmium, in an oxidized state, also reacts with C5-methylated pyrimidines. However, control of the sequence specificity of osmium complexation with DNA is still immature, and the value of the resulting complexes is unknown. We have designed a bipyridine-attached adenine derivative for sequence-specific osmium complexation. Sequence-specific osmium complexation was achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and resulted in the formation of a cross-linked structure. The interstrand cross-link clearly distinguished methylated cytosines from unmethylated cytosines and was used to quantify the degree of methylation at a specific cytosine in the genome.     

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

A separation‐free single‐base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele‐specific oligonucleotide primer (i.e., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5′‐end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite‐converted genomic DNA, and a DNA polymerase. A single base‐extended primer (i.e., SBE product) that was 5′‐Cy3‐ and 3′‐Cy5‐tagged was formed by incorporation of the Cy5‐labeled terminator into the 3′‐end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.     

A simple colorimetric detection of DNA methylation

In this work, we describe a simple colorimetric method to detect DNA methylation. Adenomatous polyposis coli (APC) with a small CpG region containing methylated cytosine (methylated APC) was synthesized and tested. Methylated APC was first captured and enriched by anti-5-methylcytosine monoclonal antibody conjugated magnetic microspheres (MMPs). Then a probe partly complementary to the APC sequence was added, resulting in the formation of DNA duplexes. The microsphere-captured probe was then released by heat denaturation and added into unmodified gold nanoparticle (AuNP) solution. Colorimetric detection was performed by salt-induced aggregation. The limit of detection is 80 fmol. Semi-quantitative analysis was done with a UV/Vis spectrophotometer by recording the absorbance of AuNP solution at 520 nm. Thus, this method provides a simple, rapid and quantitative tool for DNA methylation detection.     

Development of a body fluid identification multiplex via DNA methylation analysis

The goal of this study is to develop an epigenetic multiplex for body fluid identification based on tissue specific DNA methylation. A series of genetic loci capable of discerning the origin of DNA as coming from saliva, blood, vaginal epithelia, or semen were used for this application. The markers – BCAS4, CG06379435, VE_8, and ZC3H12D – were amplified together and then sequenced via pyrosequencing. Methylation values for cytosine guanine dinucleotide (CpG) sites at each locus were then measured across the four markers. In total, 124 samples were collected, and bisulfite modified to convert unmethylated DNA to uracil. This converted DNA was then amplified via multiplex PCR with reverse primers containing a biotin molecule. Biotinylated PCR products were then analyzed using pyrosequencing to generate a series of pyrograms containing 18 CpG sites. The percent methylation at each CpG site was determined, and then agglomerative hierarchical cluster analysis was used to create a model to indicate sample origin. Further analysis reduced the number of CpG sites required for optimal determination of body fluid type to five. This study demonstrates an efficient multiplexed body fluid identification process utilizing DNA methylation that can be easily implemented in forensic laboratories.