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<p style="text-align: center;"><strong>Make your Bisulfite conversion now in only 60 minutes !</strong></p>
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<div style="text-align: justify;" class="large-12 columns">Bisulfite modification of DNA is the most commonly used, "<strong>gold standard</strong>" method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. T<span style="font-weight: 400;">his technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at the singe nucleotide level.</span></div>
<div style="text-align: justify;" class="large-12 columns"></div>
<div style="text-align: justify;" class="large-12 columns">Various analyses can be performed on the altered sequence to retrieve this information: bisulfite sequencing, pyrosequencing, methylation-specific PCR, high resolution melting curve analysis, microarray-based approaches, and next-generation sequencing.
<h3>How it works</h3>
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected (see Figure 1).
<p class="text-center"><img src="https://www.diagenode.com/img/applications/bisulfite.png" /><br />Figure 1: Overview of bisulfite conversion of DNA</p>
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<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
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<div class="small-12 medium-4 large-4 columns text-center"><a href="../landing-pages/dna-methylation-grant-applications"><img src="https://www.diagenode.com/img/banners/banner-dna-grant.png" alt="" /></a></div>
<div style="text-align: justify;" class="small-12 medium-12 large-12 columns">
<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
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<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
</div>
</li>
</ul>
<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
</ol>
<p><span style="font-weight: 400;"> </span></p>
<div class="row">
<table>
<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
</tr>
</thead>
<tbody>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
</ul>
</td>
</tr>
</tbody>
</table>
</div>
</div>
<div class="row"></div>
</div>
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<div class="large-12 columns"></div>
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<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
<p><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/bisulfite-conversion-acgautac.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h2>Advantages</h2>
<ul class="nobullet" style="font-size: 19px;">
<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
</ul>
<h2>Downstream analysis techniques</h2>
<ul class="square">
<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
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<p><span>Fastest method for complete bisulfite conversion of DNA for methylation analysis. </span></p>
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<p><span>Ready-to-use conversion reagent is added directly to DNA. </span></p>
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<p><span>High-yield, converted DNA is ideal for PCR, MSP, array, bisulfite and Next-Gen Sequencing. </span></p>
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'description' => '<p>5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes. The ideal method for 5mC localization would be both nondestructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here we present direct methylation sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution using nanogram quantities of DNA. DM-Seq employs two key DNA-modifying enzymes: a neomorphic DNA methyltransferase and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities with deaminase-resistant adapters enables accurate detection of only 5mC via a C-to-T transition in sequencing. By comparison, we uncover a PCR-related underdetection bias with the hybrid enzymatic-chemical TET-assisted pyridine borane sequencing approach. Importantly, we show that DM-Seq, unlike bisulfite sequencing, unmasks prognostically important CpGs in a clinical tumor sample by not confounding 5mC with 5-hydroxymethylcytosine. DM-Seq thus offers an all-enzymatic, nondestructive, faithful and direct method for the reading of 5mC alone.</p>',
'date' => '2023-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37322153',
'doi' => '10.1038/s41589-023-01318-1',
'modified' => '2023-08-01 14:40:01',
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'name' => 'Transfer of blocker-based qPCR reactions for DNA methylation analysisinto a microfluidic LoC system using thermal modeling.',
'authors' => 'Kärcher J.et al.',
'description' => '<p>Changes in the DNA methylation landscape are associated with many diseases like cancer. Therefore, DNA methylation analysis is of great interest for molecular diagnostics and can be applied, e.g., for minimally invasive diagnostics in liquid biopsy samples like blood plasma. Sensitive detection of local methylation, which occurs in various cancer types, can be achieved with quantitative HeavyMethyl-PCR using oligonucleotides that block the amplification of unmethylated DNA. A transfer of these quantitative PCRs (qPCRs) into point-of-care (PoC) devices like microfluidic Lab-on-Chip (LoC) cartridges can be challenging as LoC systems show significantly different thermal properties than qPCR cyclers. We demonstrate how an adequate thermal model of the specific LoC system can help us to identify a suitable thermal profile, even for complex HeavyMethyl qPCRs, with reduced experimental effort. Using a simulation-based approach, we demonstrate a proof-of-principle for the successful LoC transfer of colorectal /-qPCR from Epi Procolon® colorectal carcinoma test, by avoidance of oligonucleotide interactions.</p>',
'date' => '2022-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36506005',
'doi' => '10.1063/5.0108374',
'modified' => '2023-03-28 09:15:30',
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'id' => '4370',
'name' => 'Development of a quantitative methylation-specific droplet digital PCRassay for detecting Dickkopf-related protein 3.',
'authors' => 'Araki K. et al.',
'description' => '<p>OBJECTIVE: The detection and monitoring of DNA methylation status in circulating tumor cell DNA (ctDNA) provides critical insights into cancer diagnosis and progression. The methylation status of the Dickkopf-related protein 3 (DKK3) promoter region is correlated with the metastasis and recurrence of multiple cancers. Thus, detecting the methylation status via non-invasive methods is essential for the diagnosis and prognosis of cancers. Using a droplet digital polymerase chain reaction approach, we have developed a highly sensitive and quantitative measurement of methylated and unmethylated DKK3 derived from circulating cell-free DNA (ccfDNA). RESULTS: We confirmed the specificity of droplet digital methylation specific polymerase chain reaction (ddMSP). We selected the optimal bisulfite conversion method using commercially available kits. We validated the ddMSP analysis system by analyzing the methylation status of genomic DNA extracted from cultured mesothelioma cells and mesothelial cells. Our system quantified approximately 30 copies of cell-free DNA per 4 mL, which is sufficient for detecting ctDNA. Finally, we quantified methylated and unmethylated DKK3 copies in ccfDNA from 21 patients with malignant mesothelioma.</p>',
'date' => '2022-05-01',
'pmid' => 'https://doi.org/10.21203%2Frs.3.rs-1323644%2Fv1',
'doi' => '10.1186/s13104-022-06056-6',
'modified' => '2022-08-04 16:03:49',
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'id' => '4293',
'name' => 'Development and inter-laboratory validation of the VISAGE enhanced toolfor age estimation from semen using quantitative DNA methylationanalysis.',
'authors' => 'Heidegger A. et al.',
'description' => '<p>The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8\% at the expected methylation level of 50\% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1\%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.</p>',
'date' => '2022-01-01',
'pmid' => 'https://doi.org/10.1016%2Fj.fsigen.2021.102596',
'doi' => '10.1016/j.fsigen.2021.102596',
'modified' => '2022-05-24 09:21:45',
'created' => '2022-05-19 10:41:50',
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(int) 4 => array(
'id' => '4042',
'name' => 'Differential epigenetic regulation between the alternative promoters,PRDM1α and PRDM1β, of the tumour suppressor gene PRDM1 in humanmultiple myeloma cells.',
'authors' => 'Romero-García, Raquel and Gómez-Jaramillo, Laura and Mateos, Rosa Maríaand Jiménez-Gómez, Gema and Pedreño-Horrillo, Nuria and Foncubierta,Esther and Rodríguez-Gutiérrez, Juan Francisco and Garzón, Sebastiánand Mora-López, Francisco and Rodríguez, Carm',
'description' => '<p>Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. The transcription factor PRDM1 is a master regulator of plasma cell development and is considered to be an oncosuppressor in several lymphoid neoplasms. The PRDM1β isoform is an alternative promoter of the PRDM1 gene that may interfere with the normal role of the PRDM1α isoform. To explain the induction of the PRDM1β isoform in MM and to offer potential therapeutic strategies to modulate its expression, we characterized the cis regulatory elements and epigenetic status of its promoter. We observed unexpected patterns of hypermethylation and hypomethylation at the PRDM1α and PRDM1β promoters, respectively, and prominent H3K4me1 and H3K9me2 enrichment at the PRDM1β promoter in non-expressing cell lines compared to PRDM1β-expressing cell lines. After treatment with drugs that inhibit DNA methylation, we were able to modify the activity of the PRDM1β promoter but not that of the PRDM1α promoter. Epigenetic drugs may offer the ability to control the expression of the PRDM1α/PRDM1β promoters as components of novel therapeutic approaches.</p>',
'date' => '2020-09-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32985591',
'doi' => '10.1038/s41598-020-72946-z',
'modified' => '2021-02-19 12:11:14',
'created' => '2021-02-18 10:21:53',
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'id' => '3874',
'name' => 'Recombination may occur in the absence of transcription in the immunoglobulin heavy chain recombination centre.',
'authors' => 'Oudinet C, Braikia FZ, Dauba A, Khamlichi AA',
'description' => '<p>Developing B cells undergo V(D)J recombination to generate a vast repertoire of Ig molecules. V(D)J recombination is initiated by the RAG1/RAG2 complex in recombination centres (RCs), where gene segments become accessible to the complex. Whether transcription is the causal factor of accessibility or whether it is a side product of other processes that generate accessibility remains a controversial issue. At the IgH locus, V(D)J recombination is controlled by Eμ enhancer, which directs the transcriptional, epigenetic and recombinational events in the IgH RC. Deletion of Eμ enhancer affects both transcription and recombination, making it difficult to conclude if Eμ controls the two processes through the same or different mechanisms. By using a mouse line carrying a CpG-rich sequence upstream of Eμ enhancer and analyzing transcription and recombination at the single-cell level, we found that recombination could occur in the RC in the absence of detectable transcription, suggesting that Eμ controls transcription and recombination through distinct mechanisms. Moreover, while the normally Eμ-dependent transcription and demethylating activities were impaired, recruitment of chromatin remodeling complexes was unaffected. RAG1 was efficiently recruited, thus compensating for the defective transcription-associated recruitment of RAG2, and providing a mechanistic basis for RAG1/RAG2 assembly to initiate V(D)J recombination.</p>',
'date' => '2020-02-22',
'pmid' => 'http://www.pubmed.gov/32086526',
'doi' => '10.1093/nar/gkaa108',
'modified' => '2020-03-20 17:40:41',
'created' => '2020-03-13 13:45:54',
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'id' => '3685',
'name' => 'Developmental regulation of DNA cytosine methylation at the immunoglobulin heavy chain constant locus.',
'authors' => 'Oudinet C, Braikia FZ, Dauba A, Santos JM, Khamlichi AA',
'description' => '<p>DNA cytosine methylation is involved in the regulation of gene expression during development and its deregulation is often associated with disease. Mammalian genomes are predominantly methylated at CpG dinucleotides. Unmethylated CpGs are often associated with active regulatory sequences while methylated CpGs are often linked to transcriptional silencing. Previous studies on CpG methylation led to the notion that transcription initiation is more sensitive to CpG methylation than transcriptional elongation. The immunoglobulin heavy chain (IgH) constant locus comprises multiple inducible constant genes and is expressed exclusively in B lymphocytes. The developmental B cell stage at which methylation patterns of the IgH constant genes are established, and the role of CpG methylation in their expression, are unknown. Here, we find that methylation patterns at most cis-acting elements of the IgH constant genes are established and maintained independently of B cell activation or promoter activity. Moreover, one of the promoters, but not the enhancers, is hypomethylated in sperm and early embryonic cells, and is targeted by different demethylation pathways, including AID, UNG, and ATM pathways. Combined, the data suggest that, rather than being prominently involved in the regulation of the IgH constant locus expression, DNA methylation may primarily contribute to its epigenetic pre-marking.</p>',
'date' => '2019-02-01',
'pmid' => 'http://www.pubmed.gov/30779742',
'doi' => '10.1371/journal.pgen.1007930',
'modified' => '2019-06-28 13:58:48',
'created' => '2019-06-21 14:55:31',
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'id' => '3295',
'name' => 'Reproducibility of methylated CpG typing with the Illumina MiSeq',
'authors' => 'Kampmann M.L. et al.',
'description' => '<div id="abst0005">
<p id="spar0015"><span><a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/dna-methylation" title="Learn more about DNA methylation">DNA methylation</a> patterns may be used for identification of body fluids and for age estimation of human individuals. We evaluated some of the challenges and pitfalls of studying <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/methylation" title="Learn more about Methylation">methylated</a> <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cpg-site" title="Learn more about CpG site">CpG sites</a>. We compared the methylated CpG analysis of two different methods 1) massively parallel <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/sequencing" title="Learn more about Sequencing">sequencing</a> (MPS) using the Illumina MiSeq and 2) the iPLEX assay on the MassARRAY</span><sup>®</sup> System. On the Illumina MiSeq, the standard deviation of the fraction of methylation was under 3% between replicates, whereas the reproducibility of the MassARRAY<sup>®</sup><span> was very difficult to achieve. We tested the <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/denaturation-biochemistry" title="Learn more about Denaturation (biochemistry)">denaturation</a> and conversion times of the Premium <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/bisulfite" title="Learn more about Bisulfite">Bisulfite</a> kit (Diagenode) in order to optimise the conversion rate and minimise DNA degradation. Finally, we tested the reproducibility of the methylation patterns using the Illumina MiSeq.</span></p>
</div>',
'date' => '2017-09-21',
'pmid' => 'http://www.sciencedirect.com/science/article/pii/S1875176817302688',
'doi' => '',
'modified' => '2017-12-04 11:01:32',
'created' => '2017-12-04 11:01:32',
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'id' => '3123',
'name' => 'Faithful SGCE imprinting in iPSC-derived cortical neurons: an endogenous cellular model of myoclonus-dystonia',
'authors' => 'Grütz K. et al.',
'description' => '<p>In neuropathology research, induced pluripotent stem cell (iPSC)-derived neurons are considered a tool closely resembling the patient brain. Albeit in respect to epigenetics, this concept has been challenged. We generated iPSC-derived cortical neurons from myoclonus-dystonia patients with mutations (W100G and R102X) in the maternally imprinted <i>ε-sarcoglycan (SGCE</i>) gene and analysed properties such as imprinting, mRNA and protein expression. Comparison of the promoter during reprogramming and differentiation showed tissue-independent differential methylation. DNA sequencing with methylation-specific primers and cDNA analysis in patient neurons indicated selective expression of the mutated paternal <i>SGCE</i> allele. While fibroblasts only expressed the ubiquitous mRNA isoform, brain-specific <i>SGCE</i> mRNA and ε-sarcoglycan protein were detected in iPSC-derived control neurons. However, neuronal protein levels were reduced in both mutants. Our phenotypic characterization highlights the suitability of iPSC-derived cortical neurons with <i>SGCE</i> mutations for myoclonus-dystonia research and, in more general terms, prompts the use of iPSC-derived cellular models to study epigenetic mechanisms impacting on health and disease.</p>',
'date' => '2017-02-03',
'pmid' => 'http://www.nature.com/articles/srep41156',
'doi' => '',
'modified' => '2017-02-15 17:20:42',
'created' => '2017-02-15 17:20:42',
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'name' => 'Regulation of Hox orthologues in the oyster Crassostrea gigas evidences a functional role for promoter DNA methylation in an invertebrate.',
'authors' => 'Saint-Carlier E, Riviere G',
'description' => '<p>DNA methylation within promoter regions (PRDM) controls vertebrate early gene transcription and thereby development, but is neglected outside this group. However, epigenetic features in the oyster Crassostrea gigas suggest functional significance of PDRM in invertebrates. To investigate this, reporter constructs containing in vitro methylated oyster Hox gene promoters were transfected into oyster embryos. The influence of in vivo methylation was studied using bisulfite sequencing and DNA methyltransferase inhibition during development. Our results demonstrate that methylation controls the transcriptional activity of the promoters investigated, unraveling a functional role for PRDM in a lophotrochozoan, an important finding regarding the evolution of epigenetic regulation.</p>',
'date' => '2015-06-04',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/25943713',
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'modified' => '2017-02-16 10:24:59',
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'name' => 'Auto Premium Bisulfite kit',
'description' => '<p>Diagenode's Premium Bisulfite Kit rapidly converts DNA through bisulfite treatment. Our conversion reagent is added directly to DNA, requires no intermediate steps, and results in high yields of DNA ready for downstream analysis methods including PCR and Next-Generation Sequencing.</p>',
'label1' => 'Characteristics',
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<p>The figure shows the conversion efficiency in TERTBS1 and CTS56 genomic regions when using 1 μg, 500ng and 100 ng of genomic DNA. Between 1 and 5 clones were analyzed for the different amounts of genomic DNA</p>
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<div style="text-align: justify;" class="large-12 columns">Bisulfite modification of DNA is the most commonly used, "<strong>gold standard</strong>" method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. T<span style="font-weight: 400;">his technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at the singe nucleotide level.</span></div>
<div style="text-align: justify;" class="large-12 columns"></div>
<div style="text-align: justify;" class="large-12 columns">Various analyses can be performed on the altered sequence to retrieve this information: bisulfite sequencing, pyrosequencing, methylation-specific PCR, high resolution melting curve analysis, microarray-based approaches, and next-generation sequencing.
<h3>How it works</h3>
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected (see Figure 1).
<p class="text-center"><img src="https://www.diagenode.com/img/applications/bisulfite.png" /><br />Figure 1: Overview of bisulfite conversion of DNA</p>
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<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
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<div class="small-12 medium-4 large-4 columns text-center"><a href="../landing-pages/dna-methylation-grant-applications"><img src="https://www.diagenode.com/img/banners/banner-dna-grant.png" alt="" /></a></div>
<div style="text-align: justify;" class="small-12 medium-12 large-12 columns">
<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
</div>
<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
</div>
</li>
</ul>
<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
</ol>
<p><span style="font-weight: 400;"> </span></p>
<div class="row">
<table>
<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
</tr>
</thead>
<tbody>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
</ul>
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<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
<p><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/bisulfite-conversion-acgautac.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h2>Advantages</h2>
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<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
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<h2>Downstream analysis techniques</h2>
<ul class="square">
<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
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<p><span>Fastest method for complete bisulfite conversion of DNA for methylation analysis. </span></p>
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<p><span>Ready-to-use conversion reagent is added directly to DNA. </span></p>
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<p><span>High-yield, converted DNA is ideal for PCR, MSP, array, bisulfite and Next-Gen Sequencing. </span></p>
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'name' => 'Direct enzymatic sequencing of 5-methylcytosine at single-baseresolution.',
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'description' => '<p>5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes. The ideal method for 5mC localization would be both nondestructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here we present direct methylation sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution using nanogram quantities of DNA. DM-Seq employs two key DNA-modifying enzymes: a neomorphic DNA methyltransferase and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities with deaminase-resistant adapters enables accurate detection of only 5mC via a C-to-T transition in sequencing. By comparison, we uncover a PCR-related underdetection bias with the hybrid enzymatic-chemical TET-assisted pyridine borane sequencing approach. Importantly, we show that DM-Seq, unlike bisulfite sequencing, unmasks prognostically important CpGs in a clinical tumor sample by not confounding 5mC with 5-hydroxymethylcytosine. DM-Seq thus offers an all-enzymatic, nondestructive, faithful and direct method for the reading of 5mC alone.</p>',
'date' => '2023-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37322153',
'doi' => '10.1038/s41589-023-01318-1',
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'name' => 'Transfer of blocker-based qPCR reactions for DNA methylation analysisinto a microfluidic LoC system using thermal modeling.',
'authors' => 'Kärcher J.et al.',
'description' => '<p>Changes in the DNA methylation landscape are associated with many diseases like cancer. Therefore, DNA methylation analysis is of great interest for molecular diagnostics and can be applied, e.g., for minimally invasive diagnostics in liquid biopsy samples like blood plasma. Sensitive detection of local methylation, which occurs in various cancer types, can be achieved with quantitative HeavyMethyl-PCR using oligonucleotides that block the amplification of unmethylated DNA. A transfer of these quantitative PCRs (qPCRs) into point-of-care (PoC) devices like microfluidic Lab-on-Chip (LoC) cartridges can be challenging as LoC systems show significantly different thermal properties than qPCR cyclers. We demonstrate how an adequate thermal model of the specific LoC system can help us to identify a suitable thermal profile, even for complex HeavyMethyl qPCRs, with reduced experimental effort. Using a simulation-based approach, we demonstrate a proof-of-principle for the successful LoC transfer of colorectal /-qPCR from Epi Procolon® colorectal carcinoma test, by avoidance of oligonucleotide interactions.</p>',
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'description' => '<p>OBJECTIVE: The detection and monitoring of DNA methylation status in circulating tumor cell DNA (ctDNA) provides critical insights into cancer diagnosis and progression. The methylation status of the Dickkopf-related protein 3 (DKK3) promoter region is correlated with the metastasis and recurrence of multiple cancers. Thus, detecting the methylation status via non-invasive methods is essential for the diagnosis and prognosis of cancers. Using a droplet digital polymerase chain reaction approach, we have developed a highly sensitive and quantitative measurement of methylated and unmethylated DKK3 derived from circulating cell-free DNA (ccfDNA). RESULTS: We confirmed the specificity of droplet digital methylation specific polymerase chain reaction (ddMSP). We selected the optimal bisulfite conversion method using commercially available kits. We validated the ddMSP analysis system by analyzing the methylation status of genomic DNA extracted from cultured mesothelioma cells and mesothelial cells. Our system quantified approximately 30 copies of cell-free DNA per 4 mL, which is sufficient for detecting ctDNA. Finally, we quantified methylated and unmethylated DKK3 copies in ccfDNA from 21 patients with malignant mesothelioma.</p>',
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'name' => 'Development and inter-laboratory validation of the VISAGE enhanced toolfor age estimation from semen using quantitative DNA methylationanalysis.',
'authors' => 'Heidegger A. et al.',
'description' => '<p>The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8\% at the expected methylation level of 50\% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1\%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.</p>',
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'pmid' => 'https://doi.org/10.1016%2Fj.fsigen.2021.102596',
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'name' => 'Differential epigenetic regulation between the alternative promoters,PRDM1α and PRDM1β, of the tumour suppressor gene PRDM1 in humanmultiple myeloma cells.',
'authors' => 'Romero-García, Raquel and Gómez-Jaramillo, Laura and Mateos, Rosa Maríaand Jiménez-Gómez, Gema and Pedreño-Horrillo, Nuria and Foncubierta,Esther and Rodríguez-Gutiérrez, Juan Francisco and Garzón, Sebastiánand Mora-López, Francisco and Rodríguez, Carm',
'description' => '<p>Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. The transcription factor PRDM1 is a master regulator of plasma cell development and is considered to be an oncosuppressor in several lymphoid neoplasms. The PRDM1β isoform is an alternative promoter of the PRDM1 gene that may interfere with the normal role of the PRDM1α isoform. To explain the induction of the PRDM1β isoform in MM and to offer potential therapeutic strategies to modulate its expression, we characterized the cis regulatory elements and epigenetic status of its promoter. We observed unexpected patterns of hypermethylation and hypomethylation at the PRDM1α and PRDM1β promoters, respectively, and prominent H3K4me1 and H3K9me2 enrichment at the PRDM1β promoter in non-expressing cell lines compared to PRDM1β-expressing cell lines. After treatment with drugs that inhibit DNA methylation, we were able to modify the activity of the PRDM1β promoter but not that of the PRDM1α promoter. Epigenetic drugs may offer the ability to control the expression of the PRDM1α/PRDM1β promoters as components of novel therapeutic approaches.</p>',
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'description' => '<p>Developing B cells undergo V(D)J recombination to generate a vast repertoire of Ig molecules. V(D)J recombination is initiated by the RAG1/RAG2 complex in recombination centres (RCs), where gene segments become accessible to the complex. Whether transcription is the causal factor of accessibility or whether it is a side product of other processes that generate accessibility remains a controversial issue. At the IgH locus, V(D)J recombination is controlled by Eμ enhancer, which directs the transcriptional, epigenetic and recombinational events in the IgH RC. Deletion of Eμ enhancer affects both transcription and recombination, making it difficult to conclude if Eμ controls the two processes through the same or different mechanisms. By using a mouse line carrying a CpG-rich sequence upstream of Eμ enhancer and analyzing transcription and recombination at the single-cell level, we found that recombination could occur in the RC in the absence of detectable transcription, suggesting that Eμ controls transcription and recombination through distinct mechanisms. Moreover, while the normally Eμ-dependent transcription and demethylating activities were impaired, recruitment of chromatin remodeling complexes was unaffected. RAG1 was efficiently recruited, thus compensating for the defective transcription-associated recruitment of RAG2, and providing a mechanistic basis for RAG1/RAG2 assembly to initiate V(D)J recombination.</p>',
'date' => '2020-02-22',
'pmid' => 'http://www.pubmed.gov/32086526',
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'name' => 'Developmental regulation of DNA cytosine methylation at the immunoglobulin heavy chain constant locus.',
'authors' => 'Oudinet C, Braikia FZ, Dauba A, Santos JM, Khamlichi AA',
'description' => '<p>DNA cytosine methylation is involved in the regulation of gene expression during development and its deregulation is often associated with disease. Mammalian genomes are predominantly methylated at CpG dinucleotides. Unmethylated CpGs are often associated with active regulatory sequences while methylated CpGs are often linked to transcriptional silencing. Previous studies on CpG methylation led to the notion that transcription initiation is more sensitive to CpG methylation than transcriptional elongation. The immunoglobulin heavy chain (IgH) constant locus comprises multiple inducible constant genes and is expressed exclusively in B lymphocytes. The developmental B cell stage at which methylation patterns of the IgH constant genes are established, and the role of CpG methylation in their expression, are unknown. Here, we find that methylation patterns at most cis-acting elements of the IgH constant genes are established and maintained independently of B cell activation or promoter activity. Moreover, one of the promoters, but not the enhancers, is hypomethylated in sperm and early embryonic cells, and is targeted by different demethylation pathways, including AID, UNG, and ATM pathways. Combined, the data suggest that, rather than being prominently involved in the regulation of the IgH constant locus expression, DNA methylation may primarily contribute to its epigenetic pre-marking.</p>',
'date' => '2019-02-01',
'pmid' => 'http://www.pubmed.gov/30779742',
'doi' => '10.1371/journal.pgen.1007930',
'modified' => '2019-06-28 13:58:48',
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<p id="spar0015"><span><a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/dna-methylation" title="Learn more about DNA methylation">DNA methylation</a> patterns may be used for identification of body fluids and for age estimation of human individuals. We evaluated some of the challenges and pitfalls of studying <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/methylation" title="Learn more about Methylation">methylated</a> <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cpg-site" title="Learn more about CpG site">CpG sites</a>. We compared the methylated CpG analysis of two different methods 1) massively parallel <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/sequencing" title="Learn more about Sequencing">sequencing</a> (MPS) using the Illumina MiSeq and 2) the iPLEX assay on the MassARRAY</span><sup>®</sup> System. On the Illumina MiSeq, the standard deviation of the fraction of methylation was under 3% between replicates, whereas the reproducibility of the MassARRAY<sup>®</sup><span> was very difficult to achieve. We tested the <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/denaturation-biochemistry" title="Learn more about Denaturation (biochemistry)">denaturation</a> and conversion times of the Premium <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/bisulfite" title="Learn more about Bisulfite">Bisulfite</a> kit (Diagenode) in order to optimise the conversion rate and minimise DNA degradation. Finally, we tested the reproducibility of the methylation patterns using the Illumina MiSeq.</span></p>
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'description' => '<p>In neuropathology research, induced pluripotent stem cell (iPSC)-derived neurons are considered a tool closely resembling the patient brain. Albeit in respect to epigenetics, this concept has been challenged. We generated iPSC-derived cortical neurons from myoclonus-dystonia patients with mutations (W100G and R102X) in the maternally imprinted <i>ε-sarcoglycan (SGCE</i>) gene and analysed properties such as imprinting, mRNA and protein expression. Comparison of the promoter during reprogramming and differentiation showed tissue-independent differential methylation. DNA sequencing with methylation-specific primers and cDNA analysis in patient neurons indicated selective expression of the mutated paternal <i>SGCE</i> allele. While fibroblasts only expressed the ubiquitous mRNA isoform, brain-specific <i>SGCE</i> mRNA and ε-sarcoglycan protein were detected in iPSC-derived control neurons. However, neuronal protein levels were reduced in both mutants. Our phenotypic characterization highlights the suitability of iPSC-derived cortical neurons with <i>SGCE</i> mutations for myoclonus-dystonia research and, in more general terms, prompts the use of iPSC-derived cellular models to study epigenetic mechanisms impacting on health and disease.</p>',
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'description' => '<p>DNA methylation within promoter regions (PRDM) controls vertebrate early gene transcription and thereby development, but is neglected outside this group. However, epigenetic features in the oyster Crassostrea gigas suggest functional significance of PDRM in invertebrates. To investigate this, reporter constructs containing in vitro methylated oyster Hox gene promoters were transfected into oyster embryos. The influence of in vivo methylation was studied using bisulfite sequencing and DNA methyltransferase inhibition during development. Our results demonstrate that methylation controls the transcriptional activity of the promoters investigated, unraveling a functional role for PRDM in a lophotrochozoan, an important finding regarding the evolution of epigenetic regulation.</p>',
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'meta_description' => 'Bisulfite modification of DNA is the most commonly used, "gold standard" method for DNA methylation studies. Since bisulfite treatment introduces specific changes in the DNA sequence depending on the methylation status of individual cytosine residues',
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<div class="large-12 columns">
<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
</div>
<div class="small-12 medium-4 large-4 columns text-center"><a href="../landing-pages/dna-methylation-grant-applications"><img src="https://www.diagenode.com/img/banners/banner-dna-grant.png" alt="" /></a></div>
<div style="text-align: justify;" class="small-12 medium-12 large-12 columns">
<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
</div>
<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
</div>
</li>
</ul>
<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
</ol>
<p><span style="font-weight: 400;"> </span></p>
<div class="row">
<table>
<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
</tr>
</thead>
<tbody>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
</ul>
</td>
</tr>
</tbody>
</table>
</div>
</div>
<div class="row"></div>
</div>
</div>
<div class="large-12 columns"></div>
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<div class="small-12 medium-8 large-8 columns"><br />
<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
<p><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/bisulfite-conversion-acgautac.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h2>Advantages</h2>
<ul class="nobullet" style="font-size: 19px;">
<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
</ul>
<h2>Downstream analysis techniques</h2>
<ul class="square">
<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
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<ul>
<li>
<p><span>Fastest method for complete bisulfite conversion of DNA for methylation analysis. </span></p>
</li>
<li>
<p><span>Ready-to-use conversion reagent is added directly to DNA. </span></p>
</li>
<li>
<p><span>High-yield, converted DNA is ideal for PCR, MSP, array, bisulfite and Next-Gen Sequencing. </span></p>
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'name' => 'Direct enzymatic sequencing of 5-methylcytosine at single-baseresolution.',
'authors' => 'Wang T. et al.',
'description' => '<p>5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes. The ideal method for 5mC localization would be both nondestructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here we present direct methylation sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution using nanogram quantities of DNA. DM-Seq employs two key DNA-modifying enzymes: a neomorphic DNA methyltransferase and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities with deaminase-resistant adapters enables accurate detection of only 5mC via a C-to-T transition in sequencing. By comparison, we uncover a PCR-related underdetection bias with the hybrid enzymatic-chemical TET-assisted pyridine borane sequencing approach. Importantly, we show that DM-Seq, unlike bisulfite sequencing, unmasks prognostically important CpGs in a clinical tumor sample by not confounding 5mC with 5-hydroxymethylcytosine. DM-Seq thus offers an all-enzymatic, nondestructive, faithful and direct method for the reading of 5mC alone.</p>',
'date' => '2023-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37322153',
'doi' => '10.1038/s41589-023-01318-1',
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'name' => 'Transfer of blocker-based qPCR reactions for DNA methylation analysisinto a microfluidic LoC system using thermal modeling.',
'authors' => 'Kärcher J.et al.',
'description' => '<p>Changes in the DNA methylation landscape are associated with many diseases like cancer. Therefore, DNA methylation analysis is of great interest for molecular diagnostics and can be applied, e.g., for minimally invasive diagnostics in liquid biopsy samples like blood plasma. Sensitive detection of local methylation, which occurs in various cancer types, can be achieved with quantitative HeavyMethyl-PCR using oligonucleotides that block the amplification of unmethylated DNA. A transfer of these quantitative PCRs (qPCRs) into point-of-care (PoC) devices like microfluidic Lab-on-Chip (LoC) cartridges can be challenging as LoC systems show significantly different thermal properties than qPCR cyclers. We demonstrate how an adequate thermal model of the specific LoC system can help us to identify a suitable thermal profile, even for complex HeavyMethyl qPCRs, with reduced experimental effort. Using a simulation-based approach, we demonstrate a proof-of-principle for the successful LoC transfer of colorectal /-qPCR from Epi Procolon® colorectal carcinoma test, by avoidance of oligonucleotide interactions.</p>',
'date' => '2022-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36506005',
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'name' => 'Development of a quantitative methylation-specific droplet digital PCRassay for detecting Dickkopf-related protein 3.',
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'description' => '<p>OBJECTIVE: The detection and monitoring of DNA methylation status in circulating tumor cell DNA (ctDNA) provides critical insights into cancer diagnosis and progression. The methylation status of the Dickkopf-related protein 3 (DKK3) promoter region is correlated with the metastasis and recurrence of multiple cancers. Thus, detecting the methylation status via non-invasive methods is essential for the diagnosis and prognosis of cancers. Using a droplet digital polymerase chain reaction approach, we have developed a highly sensitive and quantitative measurement of methylated and unmethylated DKK3 derived from circulating cell-free DNA (ccfDNA). RESULTS: We confirmed the specificity of droplet digital methylation specific polymerase chain reaction (ddMSP). We selected the optimal bisulfite conversion method using commercially available kits. We validated the ddMSP analysis system by analyzing the methylation status of genomic DNA extracted from cultured mesothelioma cells and mesothelial cells. Our system quantified approximately 30 copies of cell-free DNA per 4 mL, which is sufficient for detecting ctDNA. Finally, we quantified methylated and unmethylated DKK3 copies in ccfDNA from 21 patients with malignant mesothelioma.</p>',
'date' => '2022-05-01',
'pmid' => 'https://doi.org/10.21203%2Frs.3.rs-1323644%2Fv1',
'doi' => '10.1186/s13104-022-06056-6',
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'name' => 'Development and inter-laboratory validation of the VISAGE enhanced toolfor age estimation from semen using quantitative DNA methylationanalysis.',
'authors' => 'Heidegger A. et al.',
'description' => '<p>The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8\% at the expected methylation level of 50\% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1\%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.</p>',
'date' => '2022-01-01',
'pmid' => 'https://doi.org/10.1016%2Fj.fsigen.2021.102596',
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'id' => '4042',
'name' => 'Differential epigenetic regulation between the alternative promoters,PRDM1α and PRDM1β, of the tumour suppressor gene PRDM1 in humanmultiple myeloma cells.',
'authors' => 'Romero-García, Raquel and Gómez-Jaramillo, Laura and Mateos, Rosa Maríaand Jiménez-Gómez, Gema and Pedreño-Horrillo, Nuria and Foncubierta,Esther and Rodríguez-Gutiérrez, Juan Francisco and Garzón, Sebastiánand Mora-López, Francisco and Rodríguez, Carm',
'description' => '<p>Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. The transcription factor PRDM1 is a master regulator of plasma cell development and is considered to be an oncosuppressor in several lymphoid neoplasms. The PRDM1β isoform is an alternative promoter of the PRDM1 gene that may interfere with the normal role of the PRDM1α isoform. To explain the induction of the PRDM1β isoform in MM and to offer potential therapeutic strategies to modulate its expression, we characterized the cis regulatory elements and epigenetic status of its promoter. We observed unexpected patterns of hypermethylation and hypomethylation at the PRDM1α and PRDM1β promoters, respectively, and prominent H3K4me1 and H3K9me2 enrichment at the PRDM1β promoter in non-expressing cell lines compared to PRDM1β-expressing cell lines. After treatment with drugs that inhibit DNA methylation, we were able to modify the activity of the PRDM1β promoter but not that of the PRDM1α promoter. Epigenetic drugs may offer the ability to control the expression of the PRDM1α/PRDM1β promoters as components of novel therapeutic approaches.</p>',
'date' => '2020-09-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32985591',
'doi' => '10.1038/s41598-020-72946-z',
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'name' => 'Recombination may occur in the absence of transcription in the immunoglobulin heavy chain recombination centre.',
'authors' => 'Oudinet C, Braikia FZ, Dauba A, Khamlichi AA',
'description' => '<p>Developing B cells undergo V(D)J recombination to generate a vast repertoire of Ig molecules. V(D)J recombination is initiated by the RAG1/RAG2 complex in recombination centres (RCs), where gene segments become accessible to the complex. Whether transcription is the causal factor of accessibility or whether it is a side product of other processes that generate accessibility remains a controversial issue. At the IgH locus, V(D)J recombination is controlled by Eμ enhancer, which directs the transcriptional, epigenetic and recombinational events in the IgH RC. Deletion of Eμ enhancer affects both transcription and recombination, making it difficult to conclude if Eμ controls the two processes through the same or different mechanisms. By using a mouse line carrying a CpG-rich sequence upstream of Eμ enhancer and analyzing transcription and recombination at the single-cell level, we found that recombination could occur in the RC in the absence of detectable transcription, suggesting that Eμ controls transcription and recombination through distinct mechanisms. Moreover, while the normally Eμ-dependent transcription and demethylating activities were impaired, recruitment of chromatin remodeling complexes was unaffected. RAG1 was efficiently recruited, thus compensating for the defective transcription-associated recruitment of RAG2, and providing a mechanistic basis for RAG1/RAG2 assembly to initiate V(D)J recombination.</p>',
'date' => '2020-02-22',
'pmid' => 'http://www.pubmed.gov/32086526',
'doi' => '10.1093/nar/gkaa108',
'modified' => '2020-03-20 17:40:41',
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'name' => 'Developmental regulation of DNA cytosine methylation at the immunoglobulin heavy chain constant locus.',
'authors' => 'Oudinet C, Braikia FZ, Dauba A, Santos JM, Khamlichi AA',
'description' => '<p>DNA cytosine methylation is involved in the regulation of gene expression during development and its deregulation is often associated with disease. Mammalian genomes are predominantly methylated at CpG dinucleotides. Unmethylated CpGs are often associated with active regulatory sequences while methylated CpGs are often linked to transcriptional silencing. Previous studies on CpG methylation led to the notion that transcription initiation is more sensitive to CpG methylation than transcriptional elongation. The immunoglobulin heavy chain (IgH) constant locus comprises multiple inducible constant genes and is expressed exclusively in B lymphocytes. The developmental B cell stage at which methylation patterns of the IgH constant genes are established, and the role of CpG methylation in their expression, are unknown. Here, we find that methylation patterns at most cis-acting elements of the IgH constant genes are established and maintained independently of B cell activation or promoter activity. Moreover, one of the promoters, but not the enhancers, is hypomethylated in sperm and early embryonic cells, and is targeted by different demethylation pathways, including AID, UNG, and ATM pathways. Combined, the data suggest that, rather than being prominently involved in the regulation of the IgH constant locus expression, DNA methylation may primarily contribute to its epigenetic pre-marking.</p>',
'date' => '2019-02-01',
'pmid' => 'http://www.pubmed.gov/30779742',
'doi' => '10.1371/journal.pgen.1007930',
'modified' => '2019-06-28 13:58:48',
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'name' => 'Reproducibility of methylated CpG typing with the Illumina MiSeq',
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'description' => '<div id="abst0005">
<p id="spar0015"><span><a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/dna-methylation" title="Learn more about DNA methylation">DNA methylation</a> patterns may be used for identification of body fluids and for age estimation of human individuals. We evaluated some of the challenges and pitfalls of studying <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/methylation" title="Learn more about Methylation">methylated</a> <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cpg-site" title="Learn more about CpG site">CpG sites</a>. We compared the methylated CpG analysis of two different methods 1) massively parallel <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/sequencing" title="Learn more about Sequencing">sequencing</a> (MPS) using the Illumina MiSeq and 2) the iPLEX assay on the MassARRAY</span><sup>®</sup> System. On the Illumina MiSeq, the standard deviation of the fraction of methylation was under 3% between replicates, whereas the reproducibility of the MassARRAY<sup>®</sup><span> was very difficult to achieve. We tested the <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/denaturation-biochemistry" title="Learn more about Denaturation (biochemistry)">denaturation</a> and conversion times of the Premium <a href="http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/bisulfite" title="Learn more about Bisulfite">Bisulfite</a> kit (Diagenode) in order to optimise the conversion rate and minimise DNA degradation. Finally, we tested the reproducibility of the methylation patterns using the Illumina MiSeq.</span></p>
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'id' => '3123',
'name' => 'Faithful SGCE imprinting in iPSC-derived cortical neurons: an endogenous cellular model of myoclonus-dystonia',
'authors' => 'Grütz K. et al.',
'description' => '<p>In neuropathology research, induced pluripotent stem cell (iPSC)-derived neurons are considered a tool closely resembling the patient brain. Albeit in respect to epigenetics, this concept has been challenged. We generated iPSC-derived cortical neurons from myoclonus-dystonia patients with mutations (W100G and R102X) in the maternally imprinted <i>ε-sarcoglycan (SGCE</i>) gene and analysed properties such as imprinting, mRNA and protein expression. Comparison of the promoter during reprogramming and differentiation showed tissue-independent differential methylation. DNA sequencing with methylation-specific primers and cDNA analysis in patient neurons indicated selective expression of the mutated paternal <i>SGCE</i> allele. While fibroblasts only expressed the ubiquitous mRNA isoform, brain-specific <i>SGCE</i> mRNA and ε-sarcoglycan protein were detected in iPSC-derived control neurons. However, neuronal protein levels were reduced in both mutants. Our phenotypic characterization highlights the suitability of iPSC-derived cortical neurons with <i>SGCE</i> mutations for myoclonus-dystonia research and, in more general terms, prompts the use of iPSC-derived cellular models to study epigenetic mechanisms impacting on health and disease.</p>',
'date' => '2017-02-03',
'pmid' => 'http://www.nature.com/articles/srep41156',
'doi' => '',
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'description' => '<p>MethylTaq DNA polymerase is a high-performance Hot Start thermostable recombinant DNA polymerase. MethylTaq is an extremely robust modified Taq DNA polymerase that completely lacks any activity below 74°C thus avoiding non-specific priming at low temperature. This highly robust enzyme produces excellent results in demanding applications and it is recommended for PCR after hMeDIP (Cat. No. <span>C02010031</span>) or after Bisulfite Conversion (Cat. No. <span>C02030030</span>).</p>',
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<p>The figure shows the conversion efficiency in TERTBS1 and CTS56 genomic regions when using 1 μg, 500ng and 100 ng of genomic DNA. Between 1 and 5 clones were analyzed for the different amounts of genomic DNA</p>
<p><img src="https://www.diagenode.com/img/product/kits/auto-premium-bisulfite-clone.png" alt="Clone" /></p>',
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<div style="text-align: justify;" class="large-12 columns">Bisulfite modification of DNA is the most commonly used, "<strong>gold standard</strong>" method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. T<span style="font-weight: 400;">his technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at the singe nucleotide level.</span></div>
<div style="text-align: justify;" class="large-12 columns"></div>
<div style="text-align: justify;" class="large-12 columns">Various analyses can be performed on the altered sequence to retrieve this information: bisulfite sequencing, pyrosequencing, methylation-specific PCR, high resolution melting curve analysis, microarray-based approaches, and next-generation sequencing.
<h3>How it works</h3>
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected (see Figure 1).
<p class="text-center"><img src="https://www.diagenode.com/img/applications/bisulfite.png" /><br />Figure 1: Overview of bisulfite conversion of DNA</p>
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<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
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<div class="small-12 medium-4 large-4 columns text-center"><a href="../landing-pages/dna-methylation-grant-applications"><img src="https://www.diagenode.com/img/banners/banner-dna-grant.png" alt="" /></a></div>
<div style="text-align: justify;" class="small-12 medium-12 large-12 columns">
<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
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<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
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</li>
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<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
</ol>
<p><span style="font-weight: 400;"> </span></p>
<div class="row">
<table>
<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
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</thead>
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<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
</ul>
</td>
</tr>
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<div class="row"></div>
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<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
<p><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/bisulfite-conversion-acgautac.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h2>Advantages</h2>
<ul class="nobullet" style="font-size: 19px;">
<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
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<h2>Downstream analysis techniques</h2>
<ul class="square">
<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
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<p><span>Fastest method for complete bisulfite conversion of DNA for methylation analysis. </span></p>
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<p><span>Ready-to-use conversion reagent is added directly to DNA. </span></p>
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<p><span>High-yield, converted DNA is ideal for PCR, MSP, array, bisulfite and Next-Gen Sequencing. </span></p>
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'description' => '<p>5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes. The ideal method for 5mC localization would be both nondestructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here we present direct methylation sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution using nanogram quantities of DNA. DM-Seq employs two key DNA-modifying enzymes: a neomorphic DNA methyltransferase and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities with deaminase-resistant adapters enables accurate detection of only 5mC via a C-to-T transition in sequencing. By comparison, we uncover a PCR-related underdetection bias with the hybrid enzymatic-chemical TET-assisted pyridine borane sequencing approach. Importantly, we show that DM-Seq, unlike bisulfite sequencing, unmasks prognostically important CpGs in a clinical tumor sample by not confounding 5mC with 5-hydroxymethylcytosine. DM-Seq thus offers an all-enzymatic, nondestructive, faithful and direct method for the reading of 5mC alone.</p>',
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'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37322153',
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'name' => 'Transfer of blocker-based qPCR reactions for DNA methylation analysisinto a microfluidic LoC system using thermal modeling.',
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'description' => '<p>Changes in the DNA methylation landscape are associated with many diseases like cancer. Therefore, DNA methylation analysis is of great interest for molecular diagnostics and can be applied, e.g., for minimally invasive diagnostics in liquid biopsy samples like blood plasma. Sensitive detection of local methylation, which occurs in various cancer types, can be achieved with quantitative HeavyMethyl-PCR using oligonucleotides that block the amplification of unmethylated DNA. A transfer of these quantitative PCRs (qPCRs) into point-of-care (PoC) devices like microfluidic Lab-on-Chip (LoC) cartridges can be challenging as LoC systems show significantly different thermal properties than qPCR cyclers. We demonstrate how an adequate thermal model of the specific LoC system can help us to identify a suitable thermal profile, even for complex HeavyMethyl qPCRs, with reduced experimental effort. Using a simulation-based approach, we demonstrate a proof-of-principle for the successful LoC transfer of colorectal /-qPCR from Epi Procolon® colorectal carcinoma test, by avoidance of oligonucleotide interactions.</p>',
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'description' => '<p>OBJECTIVE: The detection and monitoring of DNA methylation status in circulating tumor cell DNA (ctDNA) provides critical insights into cancer diagnosis and progression. The methylation status of the Dickkopf-related protein 3 (DKK3) promoter region is correlated with the metastasis and recurrence of multiple cancers. Thus, detecting the methylation status via non-invasive methods is essential for the diagnosis and prognosis of cancers. Using a droplet digital polymerase chain reaction approach, we have developed a highly sensitive and quantitative measurement of methylated and unmethylated DKK3 derived from circulating cell-free DNA (ccfDNA). RESULTS: We confirmed the specificity of droplet digital methylation specific polymerase chain reaction (ddMSP). We selected the optimal bisulfite conversion method using commercially available kits. We validated the ddMSP analysis system by analyzing the methylation status of genomic DNA extracted from cultured mesothelioma cells and mesothelial cells. Our system quantified approximately 30 copies of cell-free DNA per 4 mL, which is sufficient for detecting ctDNA. Finally, we quantified methylated and unmethylated DKK3 copies in ccfDNA from 21 patients with malignant mesothelioma.</p>',
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'pmid' => 'https://doi.org/10.21203%2Frs.3.rs-1323644%2Fv1',
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'name' => 'Development and inter-laboratory validation of the VISAGE enhanced toolfor age estimation from semen using quantitative DNA methylationanalysis.',
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'description' => '<p>The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8\% at the expected methylation level of 50\% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1\%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.</p>',
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'pmid' => 'https://doi.org/10.1016%2Fj.fsigen.2021.102596',
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'name' => 'Differential epigenetic regulation between the alternative promoters,PRDM1α and PRDM1β, of the tumour suppressor gene PRDM1 in humanmultiple myeloma cells.',
'authors' => 'Romero-García, Raquel and Gómez-Jaramillo, Laura and Mateos, Rosa Maríaand Jiménez-Gómez, Gema and Pedreño-Horrillo, Nuria and Foncubierta,Esther and Rodríguez-Gutiérrez, Juan Francisco and Garzón, Sebastiánand Mora-López, Francisco and Rodríguez, Carm',
'description' => '<p>Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. The transcription factor PRDM1 is a master regulator of plasma cell development and is considered to be an oncosuppressor in several lymphoid neoplasms. The PRDM1β isoform is an alternative promoter of the PRDM1 gene that may interfere with the normal role of the PRDM1α isoform. To explain the induction of the PRDM1β isoform in MM and to offer potential therapeutic strategies to modulate its expression, we characterized the cis regulatory elements and epigenetic status of its promoter. We observed unexpected patterns of hypermethylation and hypomethylation at the PRDM1α and PRDM1β promoters, respectively, and prominent H3K4me1 and H3K9me2 enrichment at the PRDM1β promoter in non-expressing cell lines compared to PRDM1β-expressing cell lines. After treatment with drugs that inhibit DNA methylation, we were able to modify the activity of the PRDM1β promoter but not that of the PRDM1α promoter. Epigenetic drugs may offer the ability to control the expression of the PRDM1α/PRDM1β promoters as components of novel therapeutic approaches.</p>',
'date' => '2020-09-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32985591',
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<p><span>Fastest method for complete bisulfite conversion of DNA for methylation analysis. </span></p>
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<p><span>Ready-to-use conversion reagent is added directly to DNA. </span></p>
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