We offer the most comprehensive selection of DNA methylation research products to cover every step of the experiment workflow, from upstream to downstream.
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What is DNA Methylation?
DNA methylation occurs by the covalent addition of a methyl group (CH3) at the 5-carbon of the cytosine ring by DNA methyltransferases (DNMTs), resulting in 5-methylcytosine (5-mC). When a CpG island in the promoter region of a gene is methylated, expression of the gene is repressed. Several human diseases such as cancer occur when DNA methylation is not properly established and/or maintained.
DNA methylation is a dynamic and reversible process that can result in several intermediate modification forms. 5-mC converted from cytosine by DNMTs can be hydroxylated to 5-hydroxymethylcytosine (5-hmC) by TET enzymes. 5-hmC is further modified to 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) by the same TET enzymes. Analysis of DNA methylation is critical to understanding and targeting DNA methylation–associated changes, which would help identify DNA methylation markers and their biological regulation.
Which DNA Methylation Tool is Right for Your Experiment?
There are several methods available for profiling genome-wide and region-specific DNA methylation. Although cost, minimum sample input requirements, accuracy, rapidity, and throughput are important considerations, choosing the right method is most important for the success of the analysis.
Before you begin, we suggest reviewing any literature that exists related to your proposed experiment. If other researchers have already completed the legwork, you might as well take advantage of this available information as a base to build your study around. You and your team can then employ variations and unique angles to make the study different and interesting, reviewing the previously completed work carefully to identify potential obstacles or problems that the previous researchers encountered.
If you are unsure which method will work for your study, we suggest starting with a simple pre-screen of global DNA methylation and hydroxymethylation status in your samples. 1) With the use of a fast global DNA methylation or hydroxymethylation assay, you can assess whether your sample contains 5-mC or 5-hmC and how much. Then you can determine the next step and which method is appropriate to use. 2) If you find your sample contains both 5-mC and 5-hmC, oxidative bisulfite-sequencing could be used. This method can identify and distinguish 5-mC from 5-hmC on a genome-wide scale at single-base resolution. If only 5-mC is detected with little or no 5-hmC, conventional WGBS or RRBS can be performed. (3) If the sample amount is limited or single-cell level analysis is desired, post-bisulfite sequencing may meet the requirement. (4) For analyzing methylation/hydroxymethylation of specific gene panels such as a cancer gene panel or multiple genomic regions, targeted bisulfite-sequencing or targeted oxidative bisulfite-sequencing will work.
DNA samples can be prepared from cells, tissues, and body fluids and are the most commonly used starting material for the majority of DNA methylation applications and assays. Efficient, precise, and high-quality DNA isolation is a critical precursor to having a successful DNA-based experiment.
For isolating tiny amounts of DNA from microdissection samples, fresh tissue sections, formalin-fixed, and paraffin-embedded tissues, plasma, serum, body fluids, etc.
For isolation of small fragment circulating ccfDNA from plasma or serum
Methylated DNA Quantification
DNA methylation plays an essential role in normal organismal development and cellular differentiation in higher organisms. Gene expression and the development of nearly all types of cancer are also tied to DNA methylation. For example, global decrease in 5-methylcytosine content (DNA hypomethylation) is likely caused by methyl-deficiency due to a variety of environmental influences and has been proposed as a molecular marker in multiple biological processes such as cancer. Global quantification of DNA methylation is crucial for understanding the roles that gene expression and silencing play in cancer development and other diseases.
For absorbance-based quantitation of global 5-formylcytosine levels in an ELISA-like format
Bisulfite Conversion
Bisulfite and sodium bisulfite-based techniques, often known as bisulfite conversion, are common approaches for preparing DNA for gene-specific DNA methylation analysis. Treatment of DNA with bisulfite converts cytosine to uracil while leaving 5-methylcytosine intact, offering up single-nucleotide resolution information about the methylated areas of DNA. Popular methods for analyzing DNA methylation on a gene-specific basis after bisulfite treatment include bisulfite sequencing, methylation-specific PCR, and methylation-based microarrays.
Reliably detects 5hmC and 5mC simultaneously by PCR using a tiny amount of input DNA
DNA Fragmentation
Ultrasonic energy processing of DNA samples is widely accepted as a better alternative than enzymatic digestion methods. By emphasizing higher throughput processing and preventing sample contamination in our sonication technologies, our EpiSonic™ sonicators can be easily integrated into existing lab workflows and are also particularly suitable for compatibility with epigenetic and next-generation applications.
For multi-sample shearing of DNA and chromatin in next-generation sequencing applications
Methylated DNA Immunoprecipitation
Methylated DNA immunoprecipitation (meDIP) is a large-scale antibody-based technique used to enrich and capture methylated DNA fragments for gene-specific DNA methylation studies on a genome-wide scale. Following meDIP approaches, DNA methylation can be analyzed using a variety of downstream applications, including meDIP-PCR, meDIP-ChIP, and meDIP-sequencing.
For antibody-based capture of hydroxymethylated DNA fragments for microarray, PCR, or NGS applications
PCR & NGS Analysis
Next generation sequencing (NGS) technologies have increased data output volumes and lowered DNA sequencing costs beyond what is possible with standard dye-terminator methods. NGS has led to a significant advancement in understanding several epigenetic mechanisms, namely genome-wide DNA methylation and protein-DNA interaction. EpiGentek offers many inexpensive solutions for next-generation sequencing, including full package kits to conveniently construct high-quality DNA libraries from low quantity or difficult samples.
For magnetic bead separation of DNA in DNA library prep, size selection, and cleanup applications
DNA Methylation Antibodies
Well-characterized and highly validated DNA methylation antibodies and DNA demethylation antibodies may help increase researchers' understanding of DNA methylation, its involvement in diseases and cell cycle regulation, and embryonic development. DNA methylation is the most widely studied epigenetic mechanism regulating gene expression, thereby affecting cell function, while aberrant DNA methylation is prevalent in many diseases, particularly cancer.
