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  • EpiMark® Bisulfite Conversion Kit

    Description

    Genomic DNA from many organisms has modified nucleotides. In the mammalian genome, the modified base is predominately 5-methylcytosine (5-mC), which is involved in gene expression regulation, including selective inactivation of one X chromosome in females of mammalian species (1,2). Symmetrical CpG DNA methylation is heritable, but also reversible. This change of methylation status results in an enormous number of combinations of epigenetic states that can regulate gene expression. In mammalian cells, DNA methylation mainly occurs in CpG dinucleotides and is carried out by two methyltransferase enzymatic activities, namely, maintenance methylation and de novo methylation. The maintenance methyltransferase, DNMT1, is involved in the DNA methylation after every cellular DNA replication cycle. DNMT3a and DNMT3b are the de novo methyltransferases that are active in early development.

    This kit provides materials for 48 reactions. For optimal results, 50 ng–2 μg DNA is recommended.

    Kit Components: 
    Each EpiMark Bisulfite Conversion Kit contains sufficient reagents to perform the bisulfite conversion reaction of 48 samples. Store at room temperature (15–20°C). However, for long-term storage (longer than 4 weeks) Desulphonation Reaction Buffer concentrate should be stored at 2–8°C. Prepared Bisulfite Mix can be stored at -20°C for up to six months. 

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Bisulfite Mix
    Desulfonation Buffer (concentrate)10X
    Binding Buffer1X
    Wash Buffer (concentrate)5X
    Elution Buffer1X
    EpiMark spin columns with 2 ml collection tubes 50
    Solubilization Buffer

    Properties and Usage

    Materials Required but not Supplied

    Heat block or water bath (requiring temperatures of 65°C and 92°C)
    96–100% molecular biology grade ethanol
    PCR Thermal Cycler
    0.2 ml strip tubes and caps
    1.5 ml reaction tubes
    Nuclease-free Water
    Pipettors and pipette tips (for minimization of cross contamination, use aerosol
    barrier tips)

    Storage Temperature

    25°C

    Method Overview

    Method Overview

    Many tools can be used to determine the methylaton status of DNA, including antibodies, methyl binding proteins, methylation-dependent restriction enzymes, and most recently next generation sequencing. However, the most commonly used technique to date is sodium bisulfite conversion, the “gold standard” for methylation analysis (3). Incubation of the target DNA with sodium bisulfite results in conversion of all unmodified cytosines to uracils leaving the modified bases (5-mC or 5-hmC) intact. (see Figure 1). The most critical step in methylation analysis using bisulfite conversion is the complete conversion of unmodified cytosines. This is achieved by alternating cycles of thermal denaturation with incubation reactions. The protocol optimized for this kit is simple and gives consistent results.

    Figure 1. Overview of Bisulfite Conversion Reaction
    Methylated cytosines are protected and remain unchanged, while unmethylated cytosines are deaminated to uracil after treatment with sodium bisulfite.
    Figure 2. Performance evaluation of the EpiMark Bisulfite Conversion Kit.
    One μg of genomic DNA was bisulfite treated using the Epimark Bisulfite Conversion Kit, and 2 μl of eluted DNA was analyzed by end-point PCR. 388, 544, and 731 bp amplicons were amplified with primer pairs for bisulfite converted DNA (lanes 1, 3, and 5), or with primer pairs for unconverted DNA (lanes 2, 4, and 6). Epimark Hot Start Taq DNA Polymerase was used for amplification. Lanes 2, 4, and 6 clearly show no amplification product, indicating complete DNA conversion. Marker M is the 2-Log DNA Ladder (NEB #N3200).
    Figure 3. Conversion of a methylated DNA fragment.
    50 nanograms of methylated plasmid DNA was bisulfite converted and a 146 bp DNA fragment was amplified using Epimark Hot Start Taq DNA Polymerase (NEB #M0490). Amplicons were cloned, and individual clones were sequenced to determine the methylation status. Sequence aligment of 10 clones is shown to demonstrate conversion of unmethylated cytosine to thymine, while the unconverted methylated cytosine remains intact. The known methylated cytosines at the CpG site shown in red, converted cytosines marked as T, and rest of the sequence is shown as a dashed line.

    References

    1. Bestor, T.H., Verdine, G.L. (1994). Curr. Opin. Cell Biol. . 6, 380-389.
    2. Ooi, S.K., O'Donnell A.H., Bestor T.H. (2009). J. Cell Sci. 122, 2787-2791.
    3. Frommer, M., et. al. (1992). Proc. Natl. Acad. Sci. USA. 89, 1827-1831.
    4. Kumaki, Y., Oda, M. and Okano, M. (2008). Nucleic Acids Res. . 36

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    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].
    1. How should I store my bisulfite converted DNA after it is eluted from the column?
    2. Does my sample need to be free of RNA for bisulfite conversion?
    3. No PCR product is detected when I amplify more than 300 bp amplicon.
    4. Do I need extra purification of my mammalian DNA prep in order to use it in a bisulfite conversion reaction?
    5. What is the shelf life of the reagents supplied?
    6. Which DNA polymerase do you recommend for amplification of converted DNA?
    7. My thermocycler does not allow to set up the reaction volume to 140 μl, what I should do?
    1. Reagent Preparation Using EpiMark® Bisulfite Conversion Kit (E3318)
    2. End-point PCR Using EpiMark® Bisulfite Conversion Kit (E3318)
    3. Cycling Protocol Using EpiMark™ Bisulfite Conversion Kit (E3318)

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