Detection Of Global DNA Methylation Research Articles

Detection of Global DNA Methylation in Cervical Intraepithelial Neoplasia and Cancerous Lesions by Pyrosequencing and Enzyme-Linked Immunosorbent Assays.

Background:Cervical cancer is one of the most significant cancer found in women worldwide especially in developing countries. Previous reports showed that global DNA hypomethylation was correlated with various types of cancer including cervical cancer. Methods:Long interspersed nuclear element-1 (LINE1) pyrosequencing and Enzyme linked-immunosorbent assay (ELISA) assays were used for detection of global DNA methylation. The ELISA results were compared to bisulfite LINE1 pyrosequencing assay. Results:Different cervical cancer cell lines (CaSki, SiHa, HeLa, ME180, MS751, C33A) showed low global methylation percentage when compared to normal white blood cells by ELISA assay (1.47%-5.09% vs 8.20%, respectively) and by LINE1 pyrosequencing (20%-45% vs 62%, respectively). Global DNA methylation levels in cervical cancer samples were lower than precancerous lesions (Normal-CIN3) by LINE1 pyrosequencing (mean, 48.8% vs 56.9%, respectively, p<0.05) and ELISA assay (mean, 3.03% vs 3.85%, respectively, p<0.05). Conclusion:Global DNA hypomethylation was predominantly found in cervical cancer samples detected by ELISA and LINE1 pyrosequencing assays and could be used as triage tests in cervical cancer screening. ELISA assay is a suitable method for detection of global DNA methylation in large population; however, it should be further evaluated in a large clinical samples in order to be used as screening method.

Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads.

Simple SummaryGenomic profiling of cancer-derived materials in circulation has become an alternative approach for tumour genotyping. The detection of tumour origin markers such as DNA methylation in bodily fluids enables cancer screening, early-stage diagnosis and evaluation of therapy response. The development of broad platform technologies that underpin many in vitro clinical diagnostic tests has brought about a paradigm shift in cancer management and diagnosis. This study developed a multifaceted technology platform based on bioengineered polymer nanobeads for efficient capture and electrochemical detection of DNA methylation in ovarian cancer patient samples. This could be a versatile diagnostic platform for detecting numerous disease biomarkers, thus allowing several disease diagnoses.DNA methylation is a cell-type-specific epigenetic marker that is essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. It is also responsible for the pathogenesis of many diseases, including cancers. Herein, we present a simple approach for quantifying global DNA methylation in ovarian cancer patient plasma samples based on a new class of biopolymer nanobeads. Our approach utilises the immune capture of target DNA and electrochemical quantification of global DNA methylation level within the targets in a three-step strategy that involves (i) initial preparation of target single-stranded DNA (ss-DNA) from the plasma of the patients’ samples, (ii) direct adsorption of polymer nanobeads on the surface of a bare screen-printed gold electrode (SPE-Au) followed by the immobilisation of 5-methylcytosine (5mC)-horseradish peroxidase (HRP) antibody, and (iii) immune capture of target ss-DNA onto the electrode-bound PHB/5mC-HRP antibody conjugates and their subsequent qualification using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) redox cycling system. In the presence of methylated DNA, the enzymatically produced (in situ) metabolites, i.e., benzoquinone (BQ), binds irreversibly to cellular DNA resulting in the unstable formation of DNA adducts and induced oxidative DNA strand breakage. These events reduce the available BQ in the system to support the redox cycling process and sequel DNA saturation on the platform, subsequently causing high Coulombic repulsion between BQ and negatively charged nucleotide strands. Thus, the increase in methylation levels on the electrode surface is inversely proportional to the current response. The method could successfully detect as low as 5% methylation level. In addition, the assay showed good reproducibility (% RSD ≤ 5%) and specificity by analysing various levels of methylation in cell lines and plasma DNA samples from patients with ovarian cancer. We envision that our bioengineered polymer nanobeads with high surface modification versatility could be a useful alternative platform for the electrochemical detection of varying molecular biomarkers.

Application of the comet assay, micronucleus test and global DNA methylation analysis in Darevskia lizards as a sentinel organism for genotoxic monitoring of soil pollution

Widely distributed in Armenia bisexual and parthenogenetic species of Darevskia rock lizards have the potential to be considered as bioindicators of the effects of environmental pollutants. Juvenile and adult bisexual D. raddei and parthenogenetic D. armeniaca lizards were sampled from four locations with different levels of soil contamination. The comet assay, micronucleus (MN) test and global DNA methylation detection were applied in peripheral blood erythrocytes to assess genotoxic and epigenetic effects of pollutants. The concentrations of heavy metals were analyzed in the corresponding soil samples. In polluted areas levels of DNA damage were significantly higher and levels of global DNA methylation were significantly lower in both species than in reference sites. The levels of comets and global DNA methylation decreased with age but did not depend on sex. MN test did not show significant differences among localities or between sexes and age groups. The positive correlation between DNA damage and contents of Cr, Cu, As and Mo and negative correlation between global DNA methylation and contents of Cr, Cu, Zn, Mo and Pb were shown for D. raddei. The positive correlations between DNA damage and contents of Cr, Zn and Pb and negative correlations between global DNA methylation and contents of Cr, Zn, Mo, Cd and Pb were revealed for D. armeniaca. The correlation results for some metals are different in juveniles and adults. D. armeniaca showed higher sensitivity toward environmental pollution than D. raddei in their common habitat. Genetic homogeneity of parthenogenetic lizards permits to evaluate the effects of environmental pollutants independently from inter-individual genetic variation. In conclusion, the current study suggests that DNA damage and global DNA methylation may be applied to Darevskia lizards as a sentinel organism for assessing the effects of environmental pollutants.

A bisulfite treatment and PCR-free global DNA methylation detection method using electrochemical enzymatic signal engagement

In this paper we report on a bisulfite treatment and PCR amplification-free method for sensitive and selective quantifying of global DNA methylation. Our method utilizes a three-step strategy that involves (i) initial isolation and denaturation of global DNA using the standard isolation protocol and direct adsorption onto a bare gold electrode via gold-DNA affinity interaction, (ii) selective interrogation of methylation sites in adsorbed DNA via methylation-specific 5mC antibody, and (iii) subsequent signal enhancement using an electrochemical-enzymatic redox cycling reaction. In the redox cycling reaction, glucose oxidase (GOx) is used as an enzyme label, glucose as a substrate and ruthenium complex as a redox mediator. We initially investigated the enzymatic properties of GOx by varying glucose and ruthenium concentration to delineate the redox cyclic mechanism of our assay. Because of the fast electron transfer by ruthenium (Ru) complex and intrinsic signal amplification from GOx label, this method could detect as low as 5% methylation level in 50 ng of total DNA input. Moreover, the use of methylation-specific 5mC antibody conjugated GOx makes this assay relatively highly selective for DNA methylation analysis. The data obtained from the electrochemical response for different levels of methylation showed excellent interassay reproducibility of RSD (relative standard deviation) < 5% for n = 3. We believe that this inexpensive, rapid, and sensitive assay will find high relevance as an alternative method for DNA methylation analysis both in research and clinical platforms.

Porous nanozymes: the peroxidase-mimetic activity of mesoporous iron oxide for the colorimetric and electrochemical detection of global DNA methylation.

Nanomaterials (nanozymes) with peroxidase-mimetic activity have been widely used in biosensing platforms as low-cost, relatively stable and prevailing alternatives to natural enzymes. Herein, we report on the synthesis and application of the peroxidase-mimetic activity of mesoporous iron oxide (MIO) for the detection of global DNA methylation in colorectal cancer cell lines. The target DNA was extracted and denatured to get ssDNA followed by direct adsorption onto the surface of a bare screen-printed gold electrode (SPGE). A 5-methylcytosine antibody (5mC) functionalized nanomaterial (MIO-5mC) was then used to recognise the methylcytosine groups present on the SPGE. The MIO-5mC conjugates catalyse the TMB solution in the presence of hydrogen peroxide to give the colorimetric (i.e., naked-eye observation) and electrochemical detection of DNA methylation. The assay could successfully detect as low as 10% difference in the global DNA methylation level in synthetic samples and cell lines with good reproducibility and specificity (%RSD = <5%, for n = 3). This strategy avoids the use of natural enzyme horseradish peroxidase (HRP), traditional PCR based amplification and bisulfite treatment steps that are generally used in many conventional DNA methylation assays. We envisage that our assay could be a low-cost platform with great potential for genome-wide DNA methylation analysis in point-of-care applications.

Plasmonic coupling-dependent SERS of gold nanoparticles anchored on methylated DNA and detection of global DNA methylation in SERS-based platforms

Global DNA methylation is in the spotlight as a treatment and diagnostic strategy for several severe diseases. However, conventional methods entail laborious work, high costs, and high concentrations at a nanomolar range. We developed a new method based on surface-enhanced Raman spectroscopy (SERS) to detect global DNA methylation. This was chosen due to the following two reasons: (i) genomic DNA has many neighbouring methylcytosine (mCpG) sites in CpG islands, and (ii) anti-mCpG immunogold colloids are coupled to amplify plasmon coupling and then enhance SERS intensity when they bind to the mCpG sites on the genome. We first used a DNA backbone with well-defined distances between different mCpG sites to study the link between plasmon coupling and a SERS signal as a basis for plasmonic coupling-dependent SERS. The plasmon shift and SERS enhancement were monitored by plasmon coupling with different mCpG spacing on a genomic DNA backbone. Global DNA methylation was then detected via SERS in a coupled gold nanoparticle–silver nanowire (AgNW) and a simple glass-based system on a multi-channel chamber. DNA samples were simply absorbed onto a cetyltrimethylammonium bromide (CTAB)-positive-charge AgNW surface and a microscopic glass, and immunogold colloids were then used to detect the methylation status by recognizing mCpG sites. The detection limits for global methylated DNA on the platform of glass and the silver nanowire were 195 fg ml−1 and 18 fg ml−1, respectively. Moreover, the approach of aligning gold nanoparticles on methylated DNA could be applied for biomolecular templates that can assemble themselves, and a proportional correlation between the concentration and SERS intensity could allow for a quantitative assay of DNA methylation from various samples.

Epigenetics in Legg-Calvé-Perthes disease: A study of global DNA methylation.

To examine the global methylation status of DNA in blood cells of children with Legg-Calvé-Perthes disease (LCPD), since the aetiopathogenesis of LCPD remains unclear, and many factors closely associated with DNA methylation may be linked to the occurrence of LCPD. Children with LCPD and age-, sex- and body mass index-matched controls were evaluated. Methylation levels of the long interspersed nuclear element 1 (LINE-1), a biomarker of global DNA methylation, were quantified by methylation-specific polymerase chain reaction. Of 82 children with LCPD (68 male/14 female) and 120 matched controls (98 male/22 female), methylation of the LINE-1 promoter was significantly lower in patients with LCPD compared with controls. Subgroup analyses showed that methylation of the LINE-1 promoter was significantly lower in male patients with LCPD compared with male controls. No significant between-group differences were observed in female participants. Reduced global DNA methylation may be associated with increased risk of LCPD in male children. Further research is required to understand whether detection of global DNA methylation may provide a basis for clinical diagnosis and early intervention of LCPD.

Optimization of global DNA methylation measurement by LC-MS/MS and its application in lung cancer patients

Global analyses of DNA methylation contribute important insights into biology and the wide-ranging role of DNA methylation. We describe the use of online solid-phase extraction and isotope-dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of 5-methyl-2'-deoxycytidine (5-medC) and 2'-deoxycytidine (dC) in DNA. With the incorporation of isotope internal standards and online enrichment techniques, the detection limit of this method was estimated to be as low as 0.065 pg which enables human global DNA methylation detection using only picogram amounts of DNA. This method was applied to assess the optimal amounts of enzymes required for DNA digestion regarding an accurate global DNA methylation determination and completeness of digestion and to determine global methylation in human tumor adjacent lung tissue of 79 lung cancer patients. We further determined methylated (N7-methylguanine (N7-meG), O (6)-methylguanine (O (6)-meG), and N3-methyladenine (N3-meA)) and oxidized DNA lesions (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)) in lung cancer patients by LC-MS/MS. Optimization experiments revealed that dC was liberated from DNA much more readily than 5-medC by nuclease P1 and alkaline phosphatase (AP) in DNA, which could lead to an error in the global DNA methylation measurement following digestion with insufficient enzymes. Nuclease P1 showed more differential activity for 5-medC and dC than AP. Global DNA methylation levels in adenocarcinoma and squamous cell carcinoma patients were similar in the range of 3.16-4.01 %. Global DNA methylation levels were not affected by smoking and gender and were not correlated with N7-meG or 8-oxodG in lung cancer patients. Levels of O (6)-meG and N3-meA were however found to be undetectable in all lung tissue samples.

255 A novel global DNA methylation detection by fluorescence spectroscopy, a possible evaluation system of demethylating status for myelodysplastic patients

255 A novel global DNA methylation detection by fluorescence spectroscopy, a possible evaluation system of demethylating status for myelodysplastic patients

Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quantification of global DNA methylation in human sperms

Aberrant DNA methylation in human sperms has been proposed to be a possible mechanism associated with male infertility. We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method for rapid, sensitive, and specific detection of global DNA methylation level in human sperms. Multiple-reaction monitoring (MRM) mode was used in MS/MS detection for accurate quantification of DNA methylation. The intra-day and inter-day precision values of this method were within 1.50–5.70%. By using 2-deoxyguanosine as an internal standard, UPLC–MS/MS method was applied for the detection of global DNA methylation levels in three cultured cell lines. DNA methyltransferases inhibitor 5-aza-2′-deoxycytidine can significantly reduce global DNA methylation levels in treated cell lines, showing the reliability of our method. We further examined global DNA methylation levels in human sperms, and found that global methylation values varied from 3.79% to 4.65%. The average global DNA methylation level of sperm samples washed only by PBS (4.03%) was relatively lower than that of sperm samples in which abnormal and dead sperm cells were removed by density gradient centrifugation (4.25%), indicating the possible aberrant DNA methylation level in abnormal sperm cells. Clinical application of UPLC–MS/MS method in global DNA methylation detection of human sperms will be useful in human sperm quality evaluation and the study of epigenetic mechanisms responsible for male infertility.

Detection of global DNA methylation and paternally imprinted H19 gene methylation in preeclamptic placentas

Preeclampsia (PE) is a severe hypertensive disorder associated with pregnancy; despite substantial research effort in the past several years, the etiology of PE is still unclear. The role of epigenetic factors in the etiology of PE, including DNA methylation, has been poorly characterized. In the present study, we investigated global DNA methylation as well as DNA methylation of the paternally imprinted H19 gene in preeclamptic placentas. Using 5-methylcytosine immunohistochemistry and Alu and LINE-1 repeat pyrosequencing, we found that the global DNA methylation level and the DNA (cytosine-5) methyltransferase 1 mRNA level were significantly higher in the early-onset preeclamptic placentas when compared with the normal controls. Data from methylation-sensitive high resolution melting demonstrated hypermethylation of the promoter region of the H19 gene, and results of real-time PCR showed decreased mRNA expression of H19 gene in the early-onset preeclamptic placentas as compared with the normal controls. Our results suggest that abnormal DNA methylation during placentation might be involved in the pathophysiology of PE, especially early-onset preeclampsia.

Fluorescently Imaged Particle Counting Immunoassay for Sensitive Detection of DNA Modifications

Modifications of genomic DNA may change gene expression and cause adverse health effects. Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)-DNA adducts as an example. The BPDE-adducted DNA is specifically captured by immunomagnetic particles and then isolated from unmodified DNA by applying an external magnetic field. By taking advantage of the fluorescence signal amplification through multiple labeling of captured DNA by OliGreen dye, the captured BPDE-DNA adducts can be quantified by particle counting from fluorescence imaging. This clearly demonstrates that the number of fluorescently countable particles is proportional to the modification content in genomic DNA. It is interesting to note that the background fluorescence signal caused by nonspecific adsorption of OliGreen dye can be more effectively quenched than that induced by the binding of OliGreen dye to ssDNA, allowing for significant reduction in the background fluorescence and further enhancing the detection sensitivity. The developed method can detect trace BPDE-DNA adducts as low as 180 fM in the presence of 1 billion times more normal nucleotides in genomic DNA and has a dynamic range over 4 orders of magnitude. By using anti-5-methylcytosine antibody, the method is extended to the detection of global DNA methylation. With high sensitivity and specificity, this rapid and easy-to-perform analytical method for DNA modifications shows a broad spectrum of potential applications in genotoxical and epigenetic analysis.

Assessment of the genotoxicity in toad Bufo raddei exposed to petrochemical contaminants in Lanzhou Region, China

Single cell gel electrophoresis or comet assay, micronucleus (MN) test and global DNA methylation detection were used to assess the genotoxicity in toad Bufo raddei exposed to the petrochemical (mainly oil and phenol) polluted area in Lanzhou Region (LZR) comparing with a relatively unpolluted area in Liujiaxia Region (LJXR). The results from the present study indicated that DNA damage and MN frequency in toad from LZR were significantly higher than those from LJXR at the same sampling month, whereas the degree of global DNA methylation was lower, which implies that the petrochemical contaminants at environmental level in LZR were genotoxic to B. raddei. The degree of genotoxic damage was obviously related with the extent of pollution among the three sampling months in LZR. The significantly positive correlations between DNA damage and concentrations of oil and/or phenol existed in liver cells but erythrocytes, implying that liver is more suitable as a sentinel tissue for the assessment of genotoxic impact of low-level contamination. The results from both comet assay and global DNA methylation detection on liver cells showed that the genotoxicity varied significantly with oil and/or phenol concentrations, suggesting that these two methods are relatively sensitive and suitable for monitoring the genotoxicity of petrochemical pollutants on amphibians.