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DNA methylation is a fundamental mechanism in epigenetics, regulating gene expression and chromatin structure without altering the underlying DNA sequence. Epigenetic modifications like DNA methylation can influence various biological phenomena, including development, differentiation, and disease susceptibility.
DNA Methylation
The methylation of DNA is a reversible biochemical process whereby a methyl group (CH3) is added to the DNA molecule, specifically to the cytosine (C) base within the DNA sequence. The process is facilitated by DNA methyltransferases (DNMTs), which transfer CH3 from the compound S-adenosyl-L-methionine (AdoMet) to the fifth carbon position of the cytosine pyrimidine ring, resulting in the formation of 5-methylcytosine (5mC) (Figure 1). In eukaryotic cells, this occurs predominantly at cytosine bases that precede guanine bases in the DNA sequence, forming CpG dinucleotides.
Enriching 5mC DNA
Dysregulation of DNA methylation has been implicated in various human diseases, including cancer, neurodevelopmental disorders, and aging-related conditions, making the 5mC modification not only of interest in basic science research, but an important target for medicinal purposes. Thus, highly specific capture and enrichment of 5mC DNA should provide an advantage for convenient and comprehensive identification of the methylation status of normal and diseased cells that may lead to the development of new diagnostic and therapeutic methods.
Several techniques have been used for enriching methylated DNA. Histidine-tagged methyl-CpG-binding domain proteins, for example, have been employed to selectively bind 5mC DNA and isolate the methylated DNA from a pool of genomic DNA (gDNA) via nickel-coated magnetic bead separation. However, these techniques so far are considerably time consuming, labor intensive, and have low throughput. Methylated DNA Immunoprecipitation (MeDIP) offers a simpler and more cost-effective approach to 5mC DNA enrichment. MeDIP utilizes antibodies specific to 5mC to selectively immunoprecipitate methylated DNA.
How MeDIP Works
In conventional MeDIP, gDNA is first sheared into smaller fragments, usually by sonication or enzymatic digestion, to facilitate the subsequent immunoprecipitation step (Figure 2). The fragmented DNA is incubated with anti-5mC antibodies, which selectively bind to methylated cytosine residues in the DNA fragments. The antibody-DNA complexes can be captured by antibody affinity beads such as protein A/G magnetic beads or protein G agarose beads, allowing for their isolation via magnetic separation or centrifugation. The DNA is then released from the antibody and purified, and the enriched 5mC-containing fragments can be analyzed using various downstream applications, such as PCR, microarray, or NGS, to map DNA methylation patterns across the genome or at specific loci.
Simplicity, Reliability, and Consistency
A typical MeDIP protocol can be completed within 2 days. For more rapid and efficient MeDIP assays, EpigenTek offers a comprehensive suite of products for your research needs, including the popular Methylamp™ Methylated DNA Capture Kit, which uses a proprietary and unique procedure and reagent compositions to enrich 5mC DNA. The kit has the following features and advantages:
- Very efficient enrichment (>98%) of methylated DNA.
- Extremely fast and convenient procedure, which can be completed within 3 hours.
- Strip microplate format makes the assay flexible for manual or high throughput processing.
- Employs a highly specific monoclonal antibody for 5mC capture.
- Includes spin columns and collection tubes for DNA purification, saving time and reducing labor.
- High reproducibility using pre-optimized MeDIP conditions.
- Compatible with all DNA amplification-based applications.
