EpiMark^® 5-hmC and 5-mC Analysis Kit

Product Information

• 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
  E3317S     -20       Proteinase K, Molecular Biology Grade P8107AVIAL -20 1 x 0.12 ml 800 units/ml   T4 Phage β-glucosyltransferase (T4-BGT) M0357AVIAL -20 1 x 0.06 ml 10,000 units/ml   NEBuffer™ 4 B7004SVIAL -20 1 x 1.25 ml 10 X   MspI R0106AVIAL -20 1 x 0.04 ml 100,000 units/ml   HpaII R0171AVIAL -20 1 x 0.04 ml 50,000 units/ml   Unmodified Control DNA N0480AVIAL -20 1 x 0.05 ml 0.1 ng/µl   5-mC Control DNA N0481AVIAL -20 1 x 0.05 ml 0.1 ng/µl   5-hmC Control DNA N0482AVIAL -20 1 x 0.05 ml 0.1 ng/µl   Control DNA Primer Mix S0540AVIAL -20 1 x 0.04 ml 10 µM each   Uridine Diphosphate Glucose S2200SVIAL -20 1 x 0.25 ml 2 mM

• Properties & Usage

  Materials Required but not Supplied

  Heat block or water bath (suitable for temperatures of 37°C, 40°C and 95°C) PCR materials:
  

  • Locus-specific primers, flanking a CCGG site of interest
  • DNA polymerase for PCR
  • Nucleotides for PCR
  • PCR Thermal Cycler (for endpoint experiments, option IIIa)
  • Real-time PCR cycler (for quantitative experiments, option IIIb)

  0.2 ml strip tubes and caps for PCR
  1.5 ml reaction tubes
  Molecular biology grade water

• Method Overview

  Method Overview

  Step I: DNA Glucosylation Reaction with T4 β-glucosyltransferase (T4-BGT) Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent - therefore all 5-hmC will be glucosylated, unmodified or 5-mC containing DNA will not be affected.

  Step II: Restriction Endonuclease DigestionMspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation status. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI will recognize and cleave 5-mC and 5-hmC, but not 5-ghmC.

  Step III: Interrogation of the Locus by PCR as little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100–200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction (see Figure 2). Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site.

  Figure 1a: Experimental Overview

  Figure 1b: Experimental Overview

  Figure 2: Comparison of 5-hydroxymethylcytosine amounts at locus 12 in different mouse Balb/C tissue samples. (A) End-point PCR. (B) Real time PCR.

  Figure 3: High sensitivity 5-hydroxymethylcytosine detection achieved by the EpiMark kit.

• Related Products

  Companion Products

  • HpaII
  • MspI

  Materials Sold Separately

  • HpaII
  • MspI
  • NEBuffer 4
  • T4 Phage β-glucosyltransferase (T4-BGT)
  • Proteinase K, Molecular Biology Grade

• References

  1. Tahiliani, M., Koh, K.P., Shen, Y., Pastor, W.A., Bandukwala, H., Brudno, Y., Agarwal, (2009). Science 324. 930-935,
  2. Huang, Y, Pastor, W.A., Shen, Y., Tahiliani, M., Liu, D.R., Rao, A. (2010). PloS One.
  3. Kriaucionis, S. and Heintz, N. (2009). Science 324. 929-930, Epub 2009 Apr 16.

Protocols, Manuals & Usage

• Protocols

  1. Reaction Protocol for EpiMark® 5-hmC and 5-mC Analysis Kit (E3317)

• Manuals

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

  • manualE3317

• Application Notes

  • AppNote_Use_of_EpiMark_5-hmC_and_5-mC_Analysis_Kit_on_Different_Tissue_Samples

FAQs & Troubleshooting

• FAQs

  1. Does the T4-BGT show any site preference?
  2. How do I know if the glucosylation of 5-hmC in my DNA is complete?
  3. How much control DNA should I use in my qPCR?
  4. Do I need to do extra purification steps for a mammalian DNA prep in order to use it in a glucosylation reaction?

Citations & Technical Literature

• Citations

  Product Citation Tool

  Additional Citations

  • Kienhöfer S., Musheev M., Stapf U., Helm M., Schomacher L., Niehrs C., Schäfer A. (2015) GADD45a physically and functionally interacts with TET1Publication Differentiation; PubMedID: 26546041, DOI: https://www.neb.com/en/10.1016/j.diff.2015.10.003
  • Zhao C, Wang H, Zhao B, Li C, Yin R, Song M, Liu B, Liu Z, Jiang G (2014) Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine Nucleic Acids Res; 42(9), e81. PubMedID: 24682822, DOI: https://www.neb.com/en/10.1093/nar/gku216
  • Chandra S, Baribault C, Lacey M, Ehrlich M (2014) Myogenic differential methylation: diverse associations with chromatin structure Biology (Basel); 3(2), 426-51. PubMedID: 24949935, DOI: https://www.neb.com/en/10.3390/biology3020426

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]

  • E3317S_v1

• 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]

  • E3317S_v1_10026514
  • E3317S_v1_10050494
  • E3317S_v1_10060188
  • E3317S_v1_10084198
  • E3317S_v1_10104965
  • E3317S_v1_10148577
  • E3317S_v1_10171523
  • E3317S_v1_10193051
  • E3317S_v1_0021802

• Safety Data Sheets

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

  • Proteinase K, Molecular Biology Grade
  • T4 Phage β-glucosyltransferase (T4-BGT)
  • NEBuffer™ 4
  • MspI
  • HpaII
  • Unmodified Control DNA
  • 5-mC Control DNA
  • 5-hmC Control DNA
  • Control DNA Primer Mix
  • Uridine Diphosphate Glucose

• 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

  The purchase of this product conveys to the user the non-transferable right to use the purchased amount of product for Research Use Only. Commercial use of this product may require a license from New England Biolabs. Please contact busdev@neb.com for further details.