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PubMed2026

Innovative advances and clinical applications of cell-free DNA methylation detection technologies.

Advances in DNA methylation detection technologies have promoted disease-related cell-free DNA (cfDNA) analysis. CfDNA methylation profiling has the potential to serve as a promising clinical tool for early disease diagnosis. However, current detection technologies suffer from high costs, complex operational procedures, and insufficient sensitivity for low-input samples. Moreover, the definitive validation of its clinical value still awaits robust evidence from high-quality confirmatory studies. Therefore, this review begins by mapping the historical evolution of cfDNA methylation, followed by a comparison of the traditional approaches and recent breakthroughs in cfDNA methylation analysis. Specifically, this review systematically examines the two major strategies: the ones based on bisulfite-dependent DNA modification and the bisulfite-free methods, including the techniques for whole-genome methylation profiling and methods targeting specific genomic regions. Additionally, to evaluate the clinical application potential of these methods, this review comprehensively describes the details of these technologies, such as sample input requirements and sensing accuracy in detecting clinical samples. The future development of cfDNA methylation detection will focus on clinical translation, integrating technical innovations with the demands for efficient clinical diagnosis. We believe this review will help researchers select methods tailored to sample availability and clinical applicability.

Humans↗

PubMed2004

High-throughput method for detecting DNA methylation.

Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related gene may be useful for cancer diagnosis or the detection of recurrence. However, most of the studies have focused on a single gene only and gave little information about the concurrent methylation status of multiple genes. In this study, we attempted to develop a microarray method coupled with linker-PCR for detecting methylation status of multiple genes in the tumor tissue. A series of synthesized oligonucleotides were synthesised and purified to completely match with 16 investigated targets. Then they were immobilized on the aldehyde-coated glass slide to fabricate a DNA microarray for detecting methylation status of these genes. The results indicated that these genes were all methylated in the positive control. However, no methylated was found in these genes for the negative control. Only p16 and p15 genes were methylated in investigated genes for the gastric tumor tissue, whereas others were not methylated. The above results were validated by bisulfite DNA sequencing. Our experiments successfully demonstrated that the DNA microarray could be applied as a high-throughput tool to determine methylation status of the investigated genes.

Automation↗

PubMed2025

A Protocol for Detecting DNA Methylation Changes at CpG Sites of Stemness-Related Genes in Aging Stem Cells.

Aging adversely affects the self-renewal and differentiation capabilities of stem cells, which impairs tissue regeneration as well as the homeostasis. Epigenetic mechanisms, specifically DNA methylation, play a key role in the maintenance of pluripotency in stem cells and regulation of pluripotency-related gene expression. Age-related modifications in methylation patterns could influence the expression of genes critical for stem cell potency maintenance, including transcription factors Nanog and Sox2. The following chapter describes a step-by-step bisulfite sequencing protocol for detection of methylation changes in the aging stem cells and provides valuable insights into the stem cells epigenetic profile. Further, the methodology describes the steps of genomic DNA extraction, bisulfite conversion, real-time PCR amplification, and sequencing for an in-depth view of the epigenetic profile derived from aging stem cells.

DNA Methylation↗

PubMed2006

Methods for detecting DNA methylation in tumors: from bench to bedside.

Tumor-acquired changes in DNA methylation are the focus of research in an increasing number of basic, translational, and clinical laboratories around the world. In the laboratory, genome-wide technologies such as expression and DNA microarrays have been adapted to analyze patterns of DNA methylation and to screen for novel disease markers. Other technologies that are relatively inexpensive and highly sensitive such as methylation-specific PCR (MSP), or quantitative, such as quantitative MSP and pyrosequencing are widely used in retrospective studies and have potential in a diagnostic setting. In the near future, it may be possible to screen patients for common cancers using DNA methylation signatures as well as to measure patient responses to treatment, to identify patients at increased risk, or to monitor interventions designed to reduce cancer incidence. In this article, we review genome-wide and quantitative, high- resolution methods for methylation analysis that are used in the laboratory and clinic, and discuss their potential for use in a clinical setting.

Chromosome Mapping↗

PubMed2001

Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism (MSAP).

The extent of DNA methylation polymorphisms was evaluated in micropropagated banana (Musa AAA cv. 'Grand Naine') derived from either the vegetative apex of the sucker or the floral apex of the male inflorescence using the methylation-sensitive amplification polymorphism (MSAP) technique. In all, 465 fragments, each representing a recognition site cleaved by either or both of the isoschizomers were amplified using eight combinations of primers. A total of 107 sites (23%) were found to be methylated at cytosine in the genome of micropropagated banana plants. In plants micropropagated from the male inflorescence explant 14 (3%) DNA methylation events were polymorphic, while plants micropropagated from the sucker explant produced 8 (1.7%) polymorphisms. No DNA methylation polymorphisms were detected in conventionally propagated banana plants. These results demonstrated the usefulness of MSAP to detect DNA methylation events in micropropagated banana plants and indicate that DNA methylation polymorphisms are associated with micropropagation.

Journal Article↗

PubMed2025

Enhancing the sensitivity of non-invasive cervical cancer detection using CpG methylation haplotype profiling.

DNA methylation is a critical epigenetic modification that regulates gene expression and plays a significant role in cancer development. This methylation signature can be detected in cancer-derived DNA from non-invasive samples, such as plasma, urine or Pap smears. However, in early-stage cancers-when detection is most critical-the concentration of cancer DNA is often low, limiting the sensitivity of current detection methods. Traditional DNA methylation detection techniques, which rely on methylation ratio-based measurements, may obscure subtle variations in methylation patterns, further reducing detection sensitivity. In this study, we analyzed cervical scraping specimens and examined whether detecting cancer-specific methylation patterns in cervical cancer could be enhanced using a Highly Methylated Haplotype (HMH) approach. This novel approach captures highly methylated haplotypes at single-molecule resolution using next-generation sequencing, providing greater detail than conventional methods. HMHs in specific DNA regions are a hallmark of cancer and stand out in contrast to sporadic methylation commonly observed in non-cancerous tissues. We applied HMH profiling to a gene panel of four biomarkers (CA10, DPP10, FMN2, and HAS1) previously validated in cervical cancer studies. At pre-specified cutoffs (99th percentile of normals), haplotype-based scoring achieved 89.9% sensitivity for invasive cancer at high specificity (~ 94-98%), outperforming median (78.0%) and single-CpG (71.6%) methods. For clinically relevant endpoints, the combined panel detected 51-52% of CIN2 + and 66-67% of CIN3 + cases, again exceeding the performance of median- and single-CpG-based scoring methods.These findings demonstrate the potential of HMH to substantially enhance sensitivity in cervical cancer detection, offering a promising approach for non-invasive diagnostics.

Humans↗

PubMed2006

Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

Animals↗

PubMed2006

Methylation-dependent fragment separation: direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA.

Fundamental to understanding the role of cytosine (C) methylation in genomic DNA (gDNA) is the need for robust analysis methods to determine the location and degree of this modification. We report a novel method for methylation detection by denaturing capillary electrophoresis (CE) using standard fragment analysis conditions. Bisulfite treatment of gDNA will selectively deaminate C but not 5-methylcytosine (5mC). Amplicons generated from bisulfite-converted gDNA are analyzed immediately after PCR using a 6-carboxy fluorescein (6-FAM) dye-labeled primer. The amplicons from methylated and unmethylated gDNA separate based solely on base composition due to the presence of multiple C versus thymine (T) differences. By direct detection of PCR amplicons following PCR using primers that anneal independent of methylation status, the overall workflow from gDNA sample input to data analysis is relatively simple. Furthermore, the same PCR product is suitable for additional analyses such as direct sequencing, cloning and sequencing, single-base extension, and post-PCR incorporation of a modified dCTP, the latter of which allows resolution of amplicons with as little as a single C/T difference. We show the utility of this novel CE detection assay by analyzing the hypermethylated region of the fragile-X FMR1 locus.

Base Sequence↗

PubMed1995

Detection of DNA methylation in the calcitonin gene in human leukemias using differential polymerase chain reaction.

One of the earliest events in the multistep process of malignant transformation is a change in the methylation pattern of certain genes. DNA methylation is usually detected by Southern blotting after restriction digest with methylation-sensitive endonucleases. Calcitonin gene hypermethylation has been described in a variety of human malignancies including lymphomas and leukemias. Here we report a technique based on the semi-quantitative differential polymerase chain reaction (PCR) which is capable of detecting subtle changes in the methylation pattern of the human calcitonin gene. This technique is based on two principles: (i) simultaneous coamplification of the target gene (5'-region of the calcitonin gene) and a reference gene for quantitative purposes; and (ii) simultaneous coamplification of a competitor with identical primer-binding sites as the target gene to control for proper restriction digest. Using this technique, we investigated calcitonin gene methylation in a variety of human cell lines, primary leukemias and normal human blood donors. The data revealed good correlation with standard Southern blotting. Weak calcitonin gene methylation was found in all normal blood donors tested (n = 14). In contrast, strong calcitonin gene methylation was detected in most acute leukemias (five of 10 acute myeloid leukemias (AML); six of seven acute lymphoblastic leukemias (ALL)). These data show that this technique can reliably be used to quantitate gene methylation and indicate that there exists heterogeneity with regard to methylation status in different leukemias, suggesting that hypermethylation of the calcitonin gene may play a role in the transformation process of some, but not all, human leukemias. Furthermore, differential PCR may facilitate determination of calcitonin gene methylation in clinical or archival tumor samples.

Base Sequence↗

PubMed1996

Preparation of a monoclonal antibody specific for 5-methyl-2'-deoxycytidine and its application for the detection of DNA methylation levels in human peripheral blood cells.

A monoclonal antibody specific for a modified nucleoside, 5-methyl-2'-deoxycytidine (m5dCyd), was prepared using 5-methylcytidine (m5Cyd)-keyhole limpet haemocyanin (KLH) conjugate, and was characterized. Termed FMC9, the antibody reacts with m5dCyd and slightly with m5Cyd and 5-methylcytosine (m5Cyt) but not with other nucleosides tested in this investigation. FMC-9 was used in an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of m5dCyd levels. Sensitivity was in the picomole range. Methylation levels in peripheral blood cells of healthy donors were determined by inhibition ELISA. The percentage of m5dCyd in peripheral blood cells of 10 healthy donors was 5.08 +/- 0.50%. These results suggest that the inhibition ELISA using FMC9 is useful to monitor m5dCyd levels in the peripheral blood cells.

Adult↗

PubMed2002

Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives.

Methylated cytosines appear as sequence variations following bisulfite treatment and polymerase chain reaction (PCR) amplification. By using methylation-specific PCR (MSP), it is possible to detect methylated sequences in a background of unmethylated DNA with a high level of sensitivity. MSP is frequently used to identify methylated alleles in carcinogenesis, and may be combined with the TaqMan real-time PCR system, which uses fluorescence-based detection of amplification products during the amplification phase of the PCR and increases the sensitivity of detection (MethyLight). Sequences that have been incompletely converted during the bisulfite treatment are frequently coamplified during MSP, resulting in an overestimation of DNA methylation. The presence of amplified sequences originating from partially unconverted material may be determined by sequencing or by restriction digests or Southern blots of MSPs. Alternately, we have developed a method where the PCR and conversion assay are combined within a single TaqMan reaction by using an additional fluorescent probe directed against unconverted DNA (ConLight-MSP). We recommend that MSP detection always should include a step to detect unconverted DNA to avoid overestimation of the frequency or level of methylated DNA in the sample.

Blotting, Southern↗

PubMed2006

Site-specific detection of DNA methylation utilizing mCpG-SEER.

Currently there are no direct methods for the sequence-specific detection of DNA-methylation at CpG dinucleotides, which provide a possible diagnostic marker for cancer. Toward this goal, we present a methodology termed mCpG-SEquence Enabled Reassembly (mCpG-SEER) of proteins utilizing a split green fluorescent protein (GFP) tethered to specific DNA recognition elements. Our system, mCpG-SEER, employs a zinc-finger attached to one-half of GFP to target a specific sequence of dsDNA, while a methyl-CpG binding domain protein attached to the complementary half of GFP targets an adjacent methylated CpG dinucleotide site. We demonstrate that the presence of both DNA sites is necessary for the reassembly and concomitant fluorescence of the reassembled GFP. We further show that the GFP-dependent fluorescence reaches a maximum when the methyl-CpG and zinc-finger sites are separated by two base pairs and the fluorescence signal is linear to 5 pmol of methylated target DNA. Finally, the specificity of this reporter system, mCpG-SEER, was found to be >40-fold between a methylated versus a nonmethylated CpG target site.

Animals↗

PubMed2003

Sensitive detection of DNA methylation.

In recent years, many molecular biomarkers have been discovered that are capable of distinguishing tumors from normal tissue. Among the different types of markers, DNA methylation markers stand out for their potential to provide a unique combination of specificity, sensitivity, high information content, and applicability to a wide variety of clinical specimens. Methylation markers are particularly suited for situations where sensitive detection is necessary, such as when tumor DNA is either scarce or diluted by excess normal DNA. One of the most widely used methods for measuring methylation levels, methylation-specific PCR (MSP), has been proved to be a very effective tool in situations requiring sensitive detection. The addition of fluorogenic probes makes these assays more informative, quantitative, and suitable for a clinical format. The field of sensitive detection is not limited to MSP; hence, an alternative methylation-sensitive amplification is discussed. PCR-based methylation assays have been applied to the detection of tumor DNA in a variety of body fluids, including serum, plasma, urine, sputum, and lavage fluids. In many cases, the sensitivity and specificity of these detection assays has been impressive, but important technological issues remain in areas such as sample preparation, assay design, and marker selection. Once these technical concerns have been addressed, the sensitive detection of methylation will provide a powerful diagnostic and prognostic tool, especially for the early detection of preneoplastic and neoplastic lesions.

DNA Methylation↗

PubMed1991

Variable X-chromosome DNA methylation patterns detected with probe M27 beta in a series of lymphoid and myeloid malignancies.

In this study the X chromosome probe M27 beta was used to investigate DNA methylation at the DXS255 locus and hence X inactivation status and determination of tumour clonality in blood, bone marrow and biopsy tissue involved with morphologically and phenotypically defined lymphoid and myeloid disease from 14 female patients along with uninvolved bone marrow from two control individuals. Thirteen out of 16 individuals (81%) were restriction fragment length polymorphism (RFLP) heterozygous for DXS255. DNA methylation status could not be assessed in the three DXS255 homozygous individuals. In eight DXS255 heterozygous individuals clonality was clearly demonstrated using M27 beta and in six of these cases independent analysis using T cell receptor (TcR) and immunoglobulin (Ig) gene probes confirmed the presence of clonal tumour cell populations. In the two controls, polyclonality was inferred from M27 beta probe analysis. In the remaining three cases (all acute lymphoblastic leukaemia (ALL)) both DXS255 X chromosome sequences appeared to be methylated. Clonality in these cases was demonstrated by TcR or Ig monoclonal gene rearrangements. These data demonstrate the value of the M27 beta probe for determining tumour clonality in a number of cases with lymphoid and myeloid disease but indicate that there may not always be a complete correlation between DNA methylation. X inactivation status and tumour clonality in certain lymphoid neoplasms, restricting the use of this probe in clonality studies. Correlations between DNA methylation, X inactivation status and stage of normal and neoplastic T and B cell development require further investigation.

Antigens, Neoplasm↗

PubMed2005

Development and application of a multiplex PCR procedure for the detection of DNA methylation in colorectal cancer.

Cancers often exhibit aberrant methylation of gene promoter regions associated with loss of tumor suppressor and/or DNA repair gene function. Such methylation constitutes an excellent marker for the molecular detection of micro-metastases and the diagnosis of tumor recurrences. We have developed a multiplex methylation-specific PCR (MSP) procedure for rapid and simultaneous assessment of the methylation of 5 loci: the tumor suppressor genes p16INK4a, death-associated protein kinase (DAPK) and p14ARF, and the DNA repair genes hMLH1 and O6-methylguanine-DNA-methyltransferase (MGMT). This multiplex test uses one single PCR reaction and only one electrophoretic run. In 98 samples of colorectal cancer studied, methylation of MGMT, DAPK, p16, hMLH1 and p14 was present in 31, 20, 17, 16 and 14% of tumors, respectively. In 58% of the tumors at least one methylated gene was found. This multiplex MSP constitutes a simple and inexpensive method for screening of molecular signatures in colorectal cancer and can be used profitably before employing more expensive and complex techniques such as microarray testing.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Methylated DNA as a possible screening marker for neoplastic disease in several body fluids.

Early detection appears to be one of the most important approaches to reducing mortality caused by neoplasia. Changes in DNA methylation have been recognized as one of the most common molecular alterations in human tumors. Due to the ubiquity of DNA methylation changes and the possibility to detect methylated DNA in several body fluids, this specifically altered DNA may serve as a potential new screening marker for neoplastic disease.

Biomarkers, Tumor↗

PubMed2005

Association of DNA methylation of phosphoserine aminotransferase with response to endocrine therapy in patients with recurrent breast cancer.

To understand the biological basis of resistance to endocrine therapy is of utmost importance in patients with steroid hormone receptor-positive breast cancer. Not only will this allow us prediction of therapy success, it may also lead to novel therapies for patients resistant to current endocrine therapy. DNA methylation in the promoter regions of genes is a prominent epigenetic gene silencing mechanism that contributes to breast cancer biology. In the current study, we investigated whether promoter DNA methylation could be associated with resistance to endocrine therapy in patients with recurrent breast cancer. Using a microarray-based technology, the promoter DNA methylation status of 117 candidate genes was studied in a cohort of 200 steroid hormone receptor-positive tumors of patients who received the antiestrogen tamoxifen as first-line treatment for recurrent breast cancer. Of the genes analyzed, the promoter DNA methylation status of 10 genes was significantly associated with clinical outcome of tamoxifen therapy. The association of the promoter hypermethylation of the strongest marker, phosphoserine aminotransferase (PSAT1) with favorable clinical outcome was confirmed by an independent quantitative DNA methylation detection method. Furthermore, the extent of DNA methylation of PSAT1 was inversely associated with its expression at the mRNA level. Finally, also at the mRNA level, PSAT1 was a predictor of tamoxifen therapy response. Concluding, our work indicates that promoter hypermethylation and mRNA expression of PSAT1 are indicators of response to tamoxifen-based endocrine therapy in steroid hormone receptor-positive patients with recurrent breast cancer.

Adult↗

PubMed2006

Cluster analysis for DNA methylation profiles having a detection threshold.

BACKGROUND: DNA methylation, a molecular feature used to investigate tumor heterogeneity, can be measured on many genomic regions using the MethyLight technology. Due to the combination of the underlying biology of DNA methylation and the MethyLight technology, the measurements, while being generated on a continuous scale, have a large number of 0 values. This suggests that conventional clustering methodology may not perform well on this data. RESULTS: We compare performance of existing methodology (such as k-means) with two novel methods that explicitly allow for the preponderance of values at 0. We also consider how the ability to successfully cluster such data depends upon the number of informative genes for which methylation is measured and the correlation structure of the methylation values for those genes. We show that when data is collected for a sufficient number of genes, our models do improve clustering performance compared to methods, such as k-means, that do not explicitly respect the supposed biological realities of the situation. CONCLUSION: The performance of analysis methods depends upon how well the assumptions of those methods reflect the properties of the data being analyzed. Differing technologies will lead to data with differing properties, and should therefore be analyzed differently. Consequently, it is prudent to give thought to what the properties of the data are likely to be, and which analysis method might therefore be likely to best capture those properties.

Artificial Intelligence↗