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  • EpiMark® Methylated DNA Enrichment Kit

    Description

    The EpiMark® Methylated DNA Enrichment Kit will selectively bind and enrich double-stranded methyl-CpG DNA from fragmented genomic DNA with as little as 5 ng of input DNA.

    Methylated DNA is isolated from fragmented genomic DNA (5 ng–25 μg) by binding to the methyl-CpG binding domain of human MBD2 protein fused to the Fc tail of human IgG1 (MBD2-Fc), which is coupled to paramagnetic hydrophilic protein A beads (MBC2-Fc/Protein A Magnetic Bead). Two Fc domains can be bound to one site on protein A with high affinity (Kd=10-7). As the Fc fragment is a dimer, four MBD2 domains are exposed to the solvent per molecule of protein A, increasing the relative equilibrium constant 100-fold. This stable complex will selectively bind double-stranded methylated CpG containing DNA. After simple wash steps followed by magnetic capture, the enriched DNA sample is easily eluted in a small volume of nuclease-free water by incubation at 65°C. The sample is immediately ready for downstream analysis by a variety of methods including:
    • Endpoint and real-time PCR assays
    • Bisulfite conversion followed by DNA amplification
    • Cloning and sequencing
    • Direct sequencing
    • Library preparation for high-throughput sequencing
    • Labeling for DNA microarray analysis
    • Methylation-sensitive restriction enzyme-based assays
    MBD-Fc and MCIp were originally developed by Michael Rehli at the University of Regensburg to improve the sensitivity and specificity of conventional CpG binding techniques.


    Kit Components: 

    Each kit contains sufficient reagents for the enrichment of methylated DNA from up to 100 μg of fragmented input DNA. If starting with 5 ng to 10 μg of input DNA per experiment, the kit provides sufficient reagents for 25 reactions. Store at 4°C. For long-term storage > 6 months, MBD2-Fc protein should be stored at -20°C.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Fragmented HeLa DNA4100 μg/ml
    LINE Primers for Methylated Controls4100 μM
    RPL30 Primers for Non-methylated Controls4100 μM
    MirA Control Primers4100 μM
    Bind/Wash Reaction Buffer (5X)45X
    NaCl High-Salt Elution Buffer (2M)4500 mM
    MBD2a-Fc Protein-202 mg/ml
    Protein A Magnetic Beads4250 μl

    Advantages and Features

    Features


    • High-affinity binding provides greater sensitivity.
    • Elution in a small volume simplifies downstream applications.
    • Easy-to-use protocol yields enriched fractions in less than 2 hours.
    • Enriched methylated DNA fractions can be easily ligated to double-stranded adaptors for Next Generation Sequencing.
    • Highly pure product from a wide range of input DNA concentrations.

    Properties and Usage

    Materials Required but not Supplied


    50 bp DNA Ladder (NEB #N3236) 
    6-tube Magnetic Separation Rack (NEB #S1506) 
    Taq DNA Polymerase with Standard Taq Buffer (NEB #M0273) 
    Deoxynucleotide Solution Mix (NEB #N0447) 
    Nuclease-free Water

    Storage Temperature

    4°C

    Method Overview

    Method Overview


    Step I—Fragment Genomic DNA 
    DNA must be fragmented by sonication, nebulization or enzymatic treatment to 
    an average size of less than 1,000 bp. 

    Step II—Combine MBD2-Fc and Protein A Magnetic Beads in 1X Bind/Wash 
    Buffer. 
    Incubate the reaction for 15 minutes at room temperature. Wash beads two 
    times in Bind/Wash Reaction Buffer. 

    Step III—Add fragmented DNA to MBD2-Fc / Protein A Magnetic Beads. 
    Incubate the reaction for 20 minutes at room temperature. Wash beads three 
    times at room temperature for five minutes each to remove unbound DNA. 

    Step IV—Elute enriched methylated CpG DNA from beads 
    Incubate the sample at 65°C for 15 minutes in DNase-free water.

    Figure 1. Enrichment workflowFigure 1. Enrichment workflow

    Figure 2: Endpoint PCR of a methylated locus (LINE) that has been purified using the EpiMark methylated DNA Enrichment KitFigure 2: Endpoint PCR of a methylated locus (LINE) that has been purified using the EpiMark methylated DNA Enrichment Kit

    Analyzed fractions are described above gel. 




    References

    1. Cross, S.H., Charlton, J.A., Nan, X., and Bird, A.P. (1994). Nat. Genet. 6, 236-244.
    2. Fraga, M.F. et al. (2003). Nucleic Acids Res. 31, 1765-1774.
    3. Gebhard, C. et al. (2006). Nucleic Acids Res. 34, e82.
    4. Gebhard, C. et al. (2006). Cancer Research . 66, 6118-6128.
    5. Jacinto, F.V., Ballestar, E. and Esteller, M. (2008). Biotechniques . 44, 35, 37, 39 passim.
    6. Lister, R. et al. (2008). Cell. 133, 523-536.
    7. Rauch, T. and Pfeifer, G.P. (2005). Lab Invest,. 85, 1172-1180.
    8. Schilling, E. and Rehli, M. (2007). Genomics . 90, 314-323.
    9. Weber, M. et al. (2005). Nat. Genet. . 37, 853-862.

    Protocols

    1. DNA Fragmentation (E2600)
    2. Prebind MBD2-Fc to Protein A Magnetic Beads (E2600)
    3. Capture Methylated CpG DNA (E2600)
    4. Wash Off Unbound DNA (E2600)
    5. Elute Captured Methylated CpG DNA (E2600)
    6. Capture and Elute Step for Control DNA (E2600)
    7. Downstream Analysis (E2600)
    8. Protocol for use with NEBNext ChIP-Seq Library Prep Reagent Set for Illumina (E6200)

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

    Troubleshooting Guides

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.