The NEBNext® Microbiome DNA Enrichment Kit facilitates enrichment of microbial DNA from samples containing methylated host DNA (including human), by selective binding and removal of the CpG-methylated host DNA. Importantly, microbial diversity remains intact after enrichment.
In human DNA, 4–6% of cytosines are methylated, and 60–90% of these methylated cytosines are at CpG sites (1,2). In contrast, methylation at CpG sites in microbial species is rare. The NEBNext Microbiome DNA Enrichment Kit uses a simple and fast magnetic bead-based method to selectively bind and remove CpG-methylated host DNA. The method uses the MBD2-Fc protein, which is composed of the methylated CpG-specific binding protein MBD2, fused to the Fc fragment of human IgG. The Fc fragment binds readily to Protein A, enabling effective attachment to Protein A-bound magnetic beads. The MBD2 domain of this protein binds specifically and tightly to CpG methylated DNA. Application of a magnetic field then pulls out the CpG-methylated (eukaryotic) DNA, leaving the non-CpG-methylated (microbial) DNA in the supernatant (3). If desired, the host DNA captured in the magnetic bead pellet can be eluted, and a protocol is provided for this.
The NEBNext Microbiome DNA Enrichment Kit is suitable for a wide range of sample types, including samples with high levels of contaminating host DNA, (3,4) and is also effective for separation of organelle DNA (e.g. mitochondria, chloroplast) from eukaryote nuclear DNA (5). The kit is compatible with downstream applications including next generation sequencing on all platforms, qPCR and end-point PCR.
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