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, as in the MethylCollector™ MBD Capture 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.
The MethylCollector™ MBD Capture Kit utilized a His-tagged recombinant MBD2b protein which specifically binds CpG-methylated DNA fragments that have been prepared by enzymatic digestion or sonication. These protein-DNA complexes are captured with nickel-coated magnetic beads and subsequent wash steps are performed to remove fragments with little or no methylation. The methylated DNA is then eluted from the beads in the presence of Proteinase K. Due to the high efficiency of the MethylCollector MBD Capture protocol and the enormous amplification capability and specificity of PCR, analysis of the methylation status of a specific genomic DNA locus can be performed on DNA isolated from less than 170 cells cells (~1 ng DNA).
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.
- Ramsahoye, B. et al. (2000) PNAS 97, 5237-5242.