DNA Methylation Assays

MethylCollector™ Ultra is a new DNA methylation assay for the enrichment of CpG-methylated DNA. Enrichment is performed using a His-tagged recombinant methyl-binding protein 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 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.

Enriched DNA can be used in many downstream applications, such as endpoint or realtime 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.

The kit uses a combination of methyl-binding protein MBD2b with its binding partner MBD3L1, to generate higher affinity for CpG-methylated DNA than antibody-based immunoprecipitation methods.

The UnMethylCollector™ Kit is a novel DNA methylation assay for the specific isolation and enrichment of unmethylated CpG islands. UnMethylCollector™ makes it possible to identify hypomethylated promoters or to study the effects of compounds that inhibit methylation.
This simple and sensitive technique provides DNA suitable for use in many downstream applications, such as real time or endpoint PCR analysis of the methylation status of particular loci, sequencing, or amplification and labeling for microarray analysis.

The UnMethylCollector™ Kit uses a His-tagged CXXC domain from mouse Mbd1 to specifically bind unmethylated CpG islands of genomic DNA fragments that have been prepared by sonication or enzymatic digestion.

The kit provides two binding buffers; a low-salt buffer for use with samples containing less than 5 unmethylated CpG dinucleotides and a higher salt buffer for efficient binding of samples with a large number of unmethylated CpGs. The His-CXXC protein is added to the DNA fragments, and these protein-DNA complexes are captured with nickel-coated magnetic beads. Subsequent wash steps are performed using an optimized buffer and the included magnet to separate the unmethylated fragments from the rest of the genomic DNA. The unmethylated DNA is then eluted from the beads in the presence of high salt. Following clean up, the eluted DNA is ready for use in PCR analysis or other downstream applications.

Due to the high efficiency of UnMethylCollector™ 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 1600 cells (~10 ng DNA). The specificity of the CXXC domain is able to enrich for DNA fragments containing only a single unmethylated CpG.

Both assays use a fast, magnetic bead-based protocol that requires minimal starting material and can be completed in less than 3 hours.

Active Motif
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www.activemotif.com