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Epigenetics & Chromatin

Methylated DNA Enrichment

Active Motif provides a variety of kits that are designed for the detection and enrichment of DNA fragments containing methylated DNA. The resulting DNA can be used in many downstream applications, such as endpoint or real-time PCR analysis of the methylation status of particular loci in normal and diseased samples, bisulfite conversion followed by cloning and sequencing, or amplification and labeling for microarray analysis.

 

Affinity enrichment is a technique that is often used to isolate methylated DNA from the rest of the DNA population. This is usually accomplished by antibody immunoprecipitation methods or with methyl-CpG binding domain (MBD) proteins. Each method has advantages and disadvantages. The details for each are described below.

Methylated DNA Immunoprecipitation (MeDIP) is an antibody immunoprecipitation method that utilizes a 5-methylcytidine antibody to specifically recognize methylated cytosines. The MeDIP Kit requires the input DNA sample to be single-stranded in order for the 5-methylcytidine (5-mC) antibody to bind. The addition of a bridging antibody to the IP reaction significantly improves the recovery of methylated DNA. The magnetic protocol uses protein G magnetic beads and the included bar magnet for fast, simple washing and elution steps. The eluted DNA is highly enriched for DNA fragments containing 5-methylcytosine DNA methylation.

More recently, the discovery of 5-hydroxymethylcytosine (5-hmC) as a modification within genomic DNA has lead to the introduction of hMeDIP, immunoprecipitation of 5-hydroxymethylcytosine containing DNA. The hMeDIP technique uses a 5-hydroxymethylcytidine antibody to specifically recognize 5-hydroxymethylcytosine DNA. Active Motif's hMeDIP Kit differs from traditional MeDIP in that the antibody is able to efficiently bind DNA in its native double-stranded form, so denaturation is not necessary. The input DNA is incubated overnight in the presence of the 5-hmC antibody. The antibody/DNA complex is then captured using protein G magnetic beads. The beads are washed to remove any unbound DNA before the 5-hydroxymethylated DNA is eluted from the beads.

The specificity of the antibodies used enables selective enrichment of either 5-mC (MeDIP) or 5-hmC (hMeDIP) methylated DNA. MeDIP techniques are ideal for the enrichment of DNA fragments containing cytosine methylation regardless of the sequence context. While most DNA methylation in mammalian tissues occurs in a CpG context, studies have found that 15-20% of total cytosine methylation in embryonic stem (ES) cells occurs at sequences other than CpG1. For researchers interested in specific enrichment of either 5-mC and 5-hmC methylation, or researchers needing to identify total cytosine methylation, the MeDIP and hMeDIP techniques are ideal.

Another method for the enrichment of methylated DNA fragments uses recombinant methyl-binding protein MBD2b, or the MBD2b/MBD3L1 complex, as in the MethylCollector™ Ultra Kit to separate CpG methylation from the rest of the genomic DNA. The MBD enrichment technique uses the specificity of the protein binding site to selectively capture 5-methylcytosine DNA. One advantage of a methyl-CpG binding protein enrichment strategy is the input DNA sample does not need to be denatured, the protein can recognize methylated DNA in its native double-strand form. Another advantage is that the MBD protein binds only to DNA methylated in a CpG context to ensure the enrichment of methylated-CpG DNA, making this technique ideal for researchers studying CpG Islands.

MethylCollector™ Ultra is based on the Methylated CpG Island Recovery Assay (MIRA), which utilizes a His-tagged recombinant methyl-binding protein complex, MBD2b/MBD3L1, that specifically binds methylated CpGs of genomic DNA fragments that have been prepared by sonication or enzymatic digestion. Nickel-coated magnetic beads capture the protein-DNA complexes, which are then separated from the rest of the genomic DNA using the included magnet. Optimized buffers ensure that fragments with little or no methylation are removed. The methylated DNA is then eluted from the beads in the presence of Proteinase K. Following clean up, the eluted DNA is ready for use in PCR analysis or other downstream applications. MethylCollector™ Ultra can efficiently enrich for methylated DNA from as few as 170 cells (~1 ng DNA).

Because CpG islands are normally hypomethylated, the ability to enrich for unmethylated DNA fragments allows researchers to validate the methylation status of the locus of interest and identify hypomethylated DNA. While other techniques confirm methylation of CpG dinucleotides, a negative result is often designated as unmethylated DNA. With Active Motif's unique HypoMethylCollector™ Kit, unmethylated promoters can now be verified using a recombinant His-tagged CXXC protein, from the mouse Mbd1, which specifically binds unmethylated DNA. This DNA can be analyzed by either endpoint or real-time PCR, or used in a variety of other applications.

Active Motif's Hydroxymethyl Collector™ Kits are designed for the detection and enrichment of DNA fragments containing 5-hydroxymethylcytosine using a biotin-streptavidin capture method. The Hydroxymethyl Collector Kit utilizes the modification properties of β-Glucosyltransferase to specifically alter 5-hmC residues with a modified glucose. A biotin conjugate is chemcially introduced and streptavidin magnetic beads are used to enrich for DNA fragments containing 5-hydroxymethylcytosine. Enriched DNA can be used for individual gene analysis by PCR or for whole-genome analysis with microarrays or sequencing.

Regardless of the affinity enrichment method, enriched DNA can be used in many downstream applications, such as endpoint or real-time PCR analysis of the methylation status of particular loci in normal and diseased samples, bisulfite conversion followed by cloning and sequencing, or amplification and labeling for microarray analysis.


References

  1. Ramsahoye, B. et al. (2000) PNAS 97, 5237-5242.