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.
Effective enrichment of microbial genomic DNA from samples contaminating host DNA
Fast, simple protocol
Enables microbiome whole genome sequencing, even for samples with high levels of host DNA
Compatible with downstream applications including next generation sequencing on all platforms, qPCR and end point PCR
Suitable for a wide range of sample types
No requirement for live cells
Optional protocol to retain separated host DNA
Also effective for separation of organelle DNA (e.g. mitochondria, chloroplast) from eukaryote nuclear DNA
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.
Each kit contains sufficient reagents for the effective separation of CpG methylated DNA from a mixed pool containing microbial or viral DNA. If starting with 1 μg of input DNA per experiment, the volumes provided are sufficient for preparations of up to 6 reactions (NEB #E2612S) and 24 reactions (NEB #E2612L). All reactions should be stored at -20°.
Box 1: Store at -20°C.
Box 2: Store at 4°C. Do not freeze.
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George R. Feehery, Erbay Yigit, Samuel O. Oyola, Bradley W. Langhorst, Victor T. Schmidt, Fiona J. Stewart, Eileen T. Dimalanta, Linda A. Amaral-Zettler, Theodore Davis, Michael A. Quail, Sriharsa Pradhan (2013) A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA PLoS One; 8 (10), e76096 . PubMedID: 24204593
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MBD-Fc and MCiP was developed by Michael Rehli at the University of Regensburg to improve the sensitivity and specificity of conventional CpG binding techniques.
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This product and its use is the subject of one or more issued and/or pending U.S. and foreign patent applications owned by SEQUENOM, Inc. The purchase of this product from NEB or its authorized distributors conveys to the buyer the non-tranferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for profit entity). The purchase of this product does not convey a license under any method claims in the foregoing patents or patent applications directed to producing the product. The buyer cannot sell or otherwise transfer this product or its components to a third party or otherwise use the product for the following COMMERCIAL PURPOSES: (1) use of the product or its components in manufacturing; (2) use of the product or its components for therapeutic, diagnostic or prophylactic purposes in humans or animals; or (3) resale of the product or its components, whether or not such product or its components are resold for use in research.
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The MBD-Fc technology is licensed exclusively to New England Biolabs, Inc. from Sequenom, Inc.
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