EpiMark^® Methylated DNA Enrichment Kit

Product Information

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 (K_d=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.

 

Figure 1. Enrichment workflow

 

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

 

This product is related to the following categories:

Enrichment,

Analysis of DNA Methylation by Bisulfite Conversion,

Methylome Analysis Products,

DNA Methylation Analysis,

Next Generation Sequencing Library Preparation Products

• Kit Components

  The following reagents are supplied with this product:

  NEB https://www.neb.com/en/#	Component Name	Component https://www.neb.com/en/#	Stored at (°C)	Amount	Concentration
  E2600S     Multi-temperature       Bind/Wash Reaction Buffer (5X) B1032AVIAL 4 2 x 25 ml 5 X   NaCl High-Salt Elution Buffer (2M) B1033AVIAL 4 1 x 15 ml Not Applicable   LINE Primers for Methylated Controls N0485AVIAL 4 1 x 0.02 ml 100 µM   RPL30 Primers for Non-methylated Controls N0486AVIAL 4 1 x 0.02 ml 100 µM   MirA Primers for Input Control N0487AVIAL 4 1 x 0.02 ml 100 µM   MBD2a-Fc Protein M0358AVIAL -20 1 x 0.025 ml 2 mg/ml   Protein A Magnetic Beads S1426AVIAL 4 1 x 0.25 ml Not Applicable

• Properties & Usage

  Materials Required but not Supplied

  
  50 bp DNA Ladder (NEB https://www.neb.com/en/#N3236) 
  6-tube Magnetic Separation Rack (NEB https://www.neb.com/en/#S1506) 
  Taq DNA Polymerase with Standard Taq Buffer (NEB https://www.neb.com/en/#M0273) 
  Deoxynucleotide Solution Mix (NEB https://www.neb.com/en/#N0447) 
  Nuclease-free Water

• 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.

• 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 workflow

   

  Figure 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.

• Related Products

  Companion Products

  • Taq DNA Polymerase with Standard Taq Buffer
  • 50 bp DNA Ladder
  • 6-Tube Magnetic Separation Rack
  • Deoxynucleotide Solution Mix

• 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, Manuals & Usage

• Protocols

  1. DNA Fragmentation (E2600)
  2. Prebind MBD2a-Fc to Protein A Magnetic Beads (NEB https://www.neb.com/en/#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 (NEB https://www.neb.com/en/#E2600)

• Manuals

  The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

  • manualE2600

FAQs & Troubleshooting

• Troubleshooting

  • EpiMark® Methylated DNA Enrichment Kit Troubleshooting Guide

Citations & Technical Literature

• Citations

  Product Citation Tool

  Additional Citations

  • Wippermann A, Klausing S, Rupp O, Albaum SP, Büntemeyer H, Noll T, Hoffrogge R (2014) Establishment of a CpG island microarray for analyses of genome-wide DNA methylation in Chinese hamster ovary cells Appl Microbiol Biotechnol; 98(2), 579-89. PubMedID: 24146078, DOI: https://www.neb.com/en/10.1007/s00253-013-5282-2
  • McGaughey DM, Abaan HO, Miller RM, Kropp PA, Brody LC (2014) Genomics of CpG methylation in developing and developed zebrafish G3 (Bethesda); 4(5), 861-9. PubMedID: 24657902, DOI: https://www.neb.com/en/10.1534/g3.113.009514

Quality, Safety & Legal

• Quality Control Assays

  Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

• Specifications & Change Notifications

  The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

  • E2600S_v2

• Certificate of Analysis

  The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

  • E2600S_v2_10021837
  • E2600S_v2_10023769
  • E2600S_v2_10044477
  • E2600S_v2_10061145
  • E2600S_v2_10066771
  • E2600S_v2_10081384
  • E2600S_v2_10097242
  • E2600S_v2_10111295
  • E2600S_v2_10123480
  • E2600S_v2_10155004
  • E2600S_v2_10159019
  • E2600S_v2_10182938
  • E2600S_v2_10186317
  • E2600S_v2_10188842
  • E2600S_v2_10190352
  • E2600S_v2_10193050
  • E2600S_v2_10205727
  • E2600S_v2_10213986
  • E2600S_v2_10222530
  • E2600S_v2_10227034
  • E2600S_v2_10230081
  • E2600S_v2_10241298
  • E2600S_v2_10257941

• Safety Data Sheets

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

  • Bind/Wash Reaction Buffer (5X)
  • NaCl High-Salt Elution Buffer (2M)
  • LINE Primers for Methylated Controls
  • RPL30 Primers for Non-methylated Controls
  • MirA Primers for Input Control
  • MBD2a-Fc Protein
  • Protein A Magnetic Beads

• Legal and Disclaimers

  Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
  
  This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
  
  New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

  Licenses

  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.
  
  Limited Use Label License: 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-transferable 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 this 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.
  
  ACCEPTANCE OF THE FOREGOING LIMITED USE PROVISIONS: If the purchaser is not willing to accept the limitations of this limited use statement, NEB is willing to accept return of the product with a full refund. For information on purchasing a license to use this product for purposes other than those permitted above, contact the NEB Licensing Department at busdev@neb.com.
  
  The MBD-Fc technology is licensed exclusively to New England Biolabs from Sequenom, Inc.