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Results for “DNA Methylation Detection”

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At least 73 records · Page 4Linked to original sources
PubMed2004

Microarray-based method for detecting methylation changes of p16(Ink4a) gene 5'-CpG islands in gastric carcinomas.

AIM: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high-throughput approach for analyzing DNA methylation. The aim of this study was to describe a microarray-based method for detecting changes of DNA methylation in cancer. METHODS: This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Therefore, the amplified product might contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. Nine sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of p16 gene CpG islands in gastric carcinomas. The results were further validated by methylation-specific PCR (MSP). RESULTS: The experimental results showed that the microarray assay could successfully detect methylation changes of p16 gene in 18 gastric tumor samples. Moreover, it could also potentially increase the frequency of detecting p16 methylation from tumor samples than MSP. CONCLUSION: Microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci and for generating epigenetic profiles in cancer.

Base Sequence↗
PubMed2003

Liposome-mediated conformation transition of DNA detected by molecular probe: methyl green.

Recent studies have focused on the structural features of DNA-lipid assemblies. In this paper, we take methyl green (MG) as a probe molecule to detect the conformational change of DNA molecule induced by dimethyldioctadecylammonium bromide (DDAB) liposomes before the condensation process of DNA begins. DDAB-induced DNA topology changes were investigated by cyclic voltammetry (CV), circular dichroism (CD) and UV-VIS spectrometry. We find that upon binding to DNA, positively charged liposomes induce a conformational transition of DNA molecules from the native B-form to the C motif. Conformational transition in DNA results in the binding modes of MG to DNA, changing and being isolated from DNA to the solution. More stable complexes are formed between DNA and DDAB. That is also proved by the melting study of DNA.

Animals↗
PubMed2004

Denaturing gradient gel electrophoresis to detect methylation changes in DNA.

Denaturing gradient gel electrophoresis (DGGE) is a technique that fractionates DNA molecules on the basis of their melting behavior and thereby permits the separation of DNA fragments with local variations in base composition. The separation of DNA fragments by DGGE is determined by the nucleotide sequence, rather than size. This approach is effective when part of the molecule is relatively dense in G+C pairs. This separation is possible because of the pronounced drop in electrophoretic mobility in a polyacrylamide gel that occurs when a region of a DNA molecule melts, thereby forming a structure that is partly helical and partly random chain. The electrophoretic mobility of these partly melted DNA fragments is much lower than that of fully helical or fully dissociated molecules. The low residual mobility of the fragment restricts migration into more strongly denaturing regions of the gradient gel and results in focusing of the band. This property can be applied to detect the difference in melting temperature between methylated and nonmethylated DNA fragments after chemical treatment, or to enrich genomic regions in which aberrant methylation occurs. In this chapter, the application of DGGE to the analysis of genomic DNA methylation is reviewed.

Base Sequence↗
PubMed2026

DNA methylation biomarkers for early detection of ovarian cancer.

Ovarian cancer (OC) remains difficult to detect at an early stage, and current screening approaches using CA125 and transvaginal ultrasonography have not demonstrated sufficient benefit for population screening. DNA methylation is a promising biomarker class because epigenetic alterations may arise early in tumourigenesis, can be detected in circulating cell-free DNA (cfDNA), and may provide tissue-of-origin information. This review critically evaluates recent evidence on DNA methylation biomarkers for early OC detection. PubMed/MEDLINE, Web of Science, and Scopus were searched for studies published between January 2020 and September 2025, supplemented by selected earlier studies of biological or methodological relevance. Evidence was synthesised across single-gene biomarkers, multi-locus panels, genome-wide signatures, assay platforms, and machine-learning classifiers, with emphasis on early-stage performance, histological representation, comparator populations, analytical methodology, and validation design. Single-gene markers such as BRCA1, RASSF1A, OPCML, HOXA9, and HIC1 show variable performance, while multi-gene and classifier-based approaches generally provide stronger discrimination. However, many studies remain limited by retrospective case-control designs, small FIGO stage I-II subsets, predominance of serous disease, and insufficient prospective validation. Integration with CA125 may improve sensitivity but can reduce specificity, which is critical in low-prevalence screening. Clinical translation will therefore require minimal and reproducible methylation signatures, standardised low-input cfDNA workflows, rigorous external validation, and prospective longitudinal evaluation in intended-use populations.

Humans↗
PubMed2005

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.

Adenomatous Polyposis Coli Protein↗
PubMed2006

DNA methylation alterations in the pancreatic juice of patients with suspected pancreatic disease.

Molecular markers of pancreatic neoplasia could aid in the evaluation of visible pancreatic lesions and indicate neoplasia invisible to imaging. We evaluated methylation-specific PCR (MSP) assays that detect aberrantly methylated DNA for their use as markers of pancreatic neoplasia. Methylation analysis was done on pancreatic juice collected endoscopically or surgically from 155 individuals with suspected pancreatic disease: 56 patients had pancreatic ductal adenocarcinoma, 17 had intraductal papillary mucinous neoplasms, 26 had symptomatic chronic pancreatitis, 12 controls lacked evidence of pancreatic disease, and 44 were asymptomatic individuals at increased risk of developing familial pancreatic cancer undergoing screening for pancreatic neoplasia. Pancreatic juice DNA was analyzed for promoter methylation using conventional MSP assays for 17 genes. For six genes, pancreatic juice methylation was quantified using real-time quantitative MSP (QMSP; Cyclin D2, FOXE1, NPTX2, ppENK, p16, and TFPI2). Quantifying pancreatic juice methylation using QMSP with a cutoff of >1% methylated DNA could better predict pancreatic cancer than detecting methylation using conventional MSP. In the endoscopic group, 9 of 11 patients with pancreatic cancer, but none of 64 individuals without neoplasia had > or =1% methylation for two or more of the best five QMSP assays (82% sensitivity and 100% specificity; P < 0.0001). The prevalence of pancreatic juice methylation in patients with chronic pancreatitis was less than in patients with pancreatic cancer but higher than in controls and similar to high-risk individuals. The detection and quantification of aberrantly methylated DNA in pancreatic juice is a promising approach to the diagnosis of pancreatic cancer.

Adenocarcinoma↗
PubMed2006

Engineering a high-affinity methyl-CpG-binding protein.

Core members of the MBD protein family (MeCP2, MBD1, MBD2 and MBD4) share a methyl-CpG-binding domain that has a specific affinity for methylated CpG sites in double-stranded DNA. By multimerizing the MDB domain of Mbd1, we engineered a poly-MBD protein that displays methyl-CpG-specific binding in vitro with a dissociation constant that is >50-fold higher than that of a monomeric MBD. Poly-MBD proteins also localize to methylated foci in cells and can deliver a functional domain to reporter constructs in vivo. We propose that poly-MBD proteins are sensitive reagents for the detection of DNA methylation levels in isolated native DNA and for cytological detection of chromosomal CpG methylation.

Animals↗
PubMed2001

Promoter hypermethylation patterns of p16, O6-methylguanine-DNA-methyltransferase, and death-associated protein kinase in tumors and saliva of head and neck cancer patients.

Aberrant promoter hypermethylation is common in head and neck cancer and may be useful as a marker for cancer cells. We examined whether cells with tumor-specific aberrant DNA-methylation might be found in the saliva of affected patients. We tested 30 patients with primary head and neck tumors using methylation-specific PCR searching for promoter hypermethylation of the tumor suppressor gene p16 (CDKN2A), the DNA repair gene O6-methylguanine-DNA-methyltransferase (MGMT) and the putative metastasis suppressor gene death-associated protein kinase (DAP-K). Aberrant methylation of at least one of these genes was detected in 17 (56%) of 30 head and neck primary tumors; 14 (47%) of 30 at p16, 10 (33%) of 30 at Dap-K and 7 (23%) of 30 at MGMT. In 11 (65%) of 17 methylated primary tumors abnormal methylated DNA was detected in the matched saliva samples. Abnormal promoter methylation in saliva DNA was found in all tumor stages and more frequently in tumors located in the oral cavity. Moreover, none of the saliva from patients with methylation-negative tumors displayed methylation of any marker. Of 30 saliva samples from healthy control subjects (15 smokers and 15 nonsmokers), only one sample from a smoking patient was positive for DNA methylation at two target genes. Detection of aberrant promoter hypermethylation patterns of cancer-related genes in saliva of head and cancer patients is feasible and may be potentially useful for detecting and monitoring disease recurrence. Long-term longitudinal studies are needed to evaluate this approach for early detection of head and neck cancer in at-risk populations.

Apoptosis Regulatory Proteins↗
PubMed1993

Prenatal diagnosis of a hypermethylated full fragile X mutation in chorionic villi of a male fetus.

Fragile X syndrome, one of the most common human genetic diseases, is characterised by a unique genetic mechanism which involves dynamic mutation because of a heritable unstable DNA sequence and abnormal DNA methylation. Direct detection of the dynamic mutation and its methylation status at the DNA level would facilitate reliable tests for prenatal and postnatal diagnosis of the disease and for carrier detection. However, it has been suggested that DNA methylation can not be used as the basis for prenatal diagnosis as the CpG island is not always methylated in chorionic villus DNA. We report here a male fetus exhibiting both extensive somatic heterogeneity and abnormal hypermethylation of the full fragile X mutation in chorionic villus DNA as well as in fetal tissue DNA. Our results indicate that both somatic heterogeneity and hypermethylation of the full fragile X mutation are events that are clearly detectable in the 11th to 12th week of pregnancy.

Blotting, Southern↗
PubMed2003

Hypermethylation of GpG islands in the promoter region of p15(INK4b) in acute promyelocytic leukemia represses p15(INK4b) expression and correlates with poor prognosis.

We evaluated the methylation status of p15 gene in a series of 65 patients with newly diagnosed acute promyelocytic leukemia (APL) receiving homogeneous treatment. Moreover, in 32 of them, the methylation status of p15 gene was correlated to the p15 m-RNA expression. In total, 31 patients had no p15 methylation (U group). An abnormal methylation pattern was found in 34 patients: in seven of these patients only methylated DNA was detected (M group), while in the remaining 27 patients (M/U group), both methylated and unmethylated DNA were amplified. Patients from M group showed a higher incidence of relapses and a lower disease-free survival (DSF) with respect to patients from U and M/U groups (29, 64 and 79% at 5 years for M, U/M and U patients, respectively, P=0.03), while p15 methylation had no impact on overall survival. The p15 expression was detectable in all patients with unmethylated DNA, in none of patients with fully methylated DNA and in 60% of patients with partially methylated DNA. The DFS estimate at 5 years for p15-negative patients was significantly lower than that of p15-positive patients (P=0.03). These data confirm that the presence of p15 methylation negatively influences the prognosis of APL, mainly when it represses the p15 gene transcription.

Adult↗
PubMed2003

Stealth technology: how Epstein-Barr virus utilizes DNA methylation to cloak itself from immune detection.

Epstein-Barr virus (EBV) is a large lymphotrophic DNA virus that establishes life-long residency in the infected host and is associated with a number of human tumors. The EBV genome encodes proteins essential for persistence, an oncoprotein, and proteins that render it vulnerable to the host's immune system; therefore, EBV gene transcription is tightly regulated. One critically important regulatory mechanism utilized by EBV is DNA methylation. Methylation of cytosines within CpG dinucleotides at promoter regions is important for gene silencing and genome integrity. Although most parasitic elements are methylated in mammalian cells never to be reactivated again, EBV has evolved to utilize DNA methylation to maximize persistence and cloak itself from immune detection. EBV's reliance on DNA methylation also provides a unique therapeutic strategy for the treatment of EBV-associated tumors. DNA demethylating agents are capable of reactivating transcription of highly immunogenic viral proteins, rendering tumor cells susceptible to killing by the host immune system, and inducing the viral lytic cycle which culminates in cell lysis.

Binding Sites↗
PubMed1985

DNA methylation at asymmetric sites is associated with numerous transition mutations.

We describe two unusual 5S RNA regions from Neurospora crassa that are tightly linked. Sequence analysis suggests that these genes or pseudogenes, which we designate zeta (zeta) and eta (eta), arose by a 794-base-pair tandem duplication followed by hundreds of exclusively cytosine to thymine mutations. The duplication was most likely generated by nonhomologous recombination involving a DNA segment having a striking purine-pyrimidine strand asymmetry. Restriction analysis of genomic DNA from tissue grown in the presence or absence of 5-azacytidine indicates that many, and perhaps all, cytosines in the duplicated region are methylated in most cells. This is in contrast to the situation typically observed in eukaryotes, where 5-methylcytosine is found only at positions one or two nucleotides preceding guanine residues. No DNA methylation was detected in the unique DNA flanking the zeta-eta duplication. Thus the "signal" for methylation may be the duplication itself. The numerous transition mutations in this region probably occurred by deamination of 5-methylcytosines. Our results suggest that DNA methylation can have important evolutionary consequences in eukaryotes.

5-Methylcytosine↗
PubMed2004

A real-time PCR assay for DNA-methylation using methylation-specific blockers.

DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.

Adenocarcinoma↗
PubMed2005

Identification of epigenetic aberrant promoter methylation in serum DNA is useful for early detection of lung cancer.

PURPOSE: The purpose of this study is to evaluate the usefulness of serum DNA methylation of five tumor suppressor genes for early detection of lung cancer. EXPERIMENTAL DESIGN: Methylation status in serum DNA from 200 patients undergoing bronchofiberscopic examination for abnormal findings on chest radiograph detected by lung cancer screening or surveillance was examined using methylation-specific PCR. RESULTS: Ninety-one patients were given a pathologic diagnosis of lung cancer, 9 other malignant diseases, and 100 nonmalignant pulmonary diseases. In patients with lung cancer, methylation was detected in 18.7% for MGMT, 15.4% for p16(INK4a), 12.1% for RASSF1A, 11.0% for DAPK, and 6.6% for RAR-beta, which was higher compared with that in patients with nonmalignant diseases. Age and smoking status seemed to associate with methylation status. Sensitivity, specificity, and predictive value of methylation in at least one gene for diagnosis of lung cancer were 49.5%, 85.0%, and 75.0%, respectively. Adjusted odds ratio (95% confidence interval) for having lung cancer was 5.28 (2.39-11.7) for patients with methylation in one gene and 5.89 (1.53-22.7) for those with methylation in two or more genes. It is of note that methylation was identified in 50.9% of stage I lung cancer patients, whereas serum protein tumor markers were positive in 11.3% of them. CONCLUSIONS: These results suggest that identification of promoter methylation of tumor suppressor genes in serum DNA could be useful for early detection of lung cancer.

Adult↗
PubMed1982

Decreased viscosity of rat-liver DNA treated by 3'-methyl-4-dimethylaminoazobenzene, detected with a new viscometric approach.

DNA damage induced in vivo by 3'-methyl-4-dimethylaminoazobenzene (3'CH3DAB) was investigated with 2 differently sensitive techniques: the alkaline elution assay and the viscometric measurement of DNA damage. 3'CH3DAB appeared to be falsely negative with the alkaline elution assay, whereas with the viscometric approach, which is about 30-50 times more sensitive, it appeared positive, and the DNA damage was dose-dependent.

Animals↗