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  • CpG Methyltransferase (M.SssI)

    Description

    The CpG Methyltransferase, M.SssI, methylates all cytosine residues (C5) within the double-stranded dinucleotide recognition sequence 5'...CG...3' (1).

    Product Source

    The CpG Methyltransferase(M.SssI) is isolated from a strain of E. coli. which contains the Methyltransferase gene from Spiroplasma sp. strain MQ1 (2,3).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 2-2010X
    S-adenosylmethionine (SAM)-2032 mM

    Advantages and Features

    Applications

    • Blocking restriction endonuclease cleavage
    • Studying of CpG methylation-dependent gene expression
    • Probing sequence-specific contacts within the major groove of DNA
    • Altering the physical properties of DNA
    • Uniform [3H]-labeling of DNA
    • Decreasing the number of sites cut by restriction endonucleases, yielding an apparent increase in specificity

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to protect 1 μg of λ DNA in a total reaction volume of 20 μl in 1 hour at 37°C against cleavage by BstUI restriction endonuclease.

    Reaction Conditions

    1X NEBuffer 2
    Supplement with 160 μM S-adenosylmethionine (SAM)
    Incubate at 37°C

    1X NEBuffer 2:
    50 mM NaCl
    10 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Functional Test (Methyltransferase):

      The Methyltransferase is tested in a reaction with Lambda DNA and SAM, followed by a second reaction with the appropriate restriction enzyme. After incubation the extent of methylation is determined by agarose gel electrophoresis.

    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • Physical Purity (HPLC):
      The purity of the product is determined by HPLC analysis.

    Notes

    1. Methylation can be optimized by using fresh SAM.
    2. MgCl2 is not required as a cofactor. In the presence of Mg2+, methylation by M.Sss I becomes distributive rather than processive and also exhibits topoisomerase activity (4).
    3. A buffer containing 50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM EDTA, 160 µM S-adenosylmethionine may be substituted for NEBuffer 2.
    4. Adding more SAM after 4 hours can improve results. Methylation reactions, however are greatly affected by S-adenosylhomocysteine (SAH)(11). SAH, by product of the methylation reaction binds more tightly to methylases than does SAM. Inhibition by SAH greatly reduces the reaction rate. Using more enzyme for less time may improve methylation.
      • This CPG Methyltransferase may be useful for studying the function of cytosine methylation in higher eukaryotes as its specificity mimics the pattern of modification found in their genomes (5). In contrast to the mammalian enzymes (6,7), both unmethylated and hemi-methylated DNA substrates are methylated with equal efficiency by the CpG Methyltransferase(2), making it a more useful tool for modifying DNA. 
      • The CpG Methyltransferase can be used to block cleavage by a variety of restriction endonucleases whose recognition sites either contain the sequence CG, or overlap the dinucleotide. It should be noted that DNAs methylated by the CpG Methyltransferase are subject to Mcr and Mrr restriction in E. coli, and thus should be transformed into Mcr- Mrr- E. coli strains. 
      • Methylation at cytosine residues has also been shown to affect the physical properties of DNA, including lowering the free energy of Z-DNA formation (8), increasing the helical pitch of DNA (6), and altering the kinetics of cruciform extrusion (9). Positions of 5-methylcytosine can be identified due to decreased reactivity to hydrazine in chemical sequencing protocols (10). 
      • The high density of CpG dinucleotides in DNA substrates should be taken into account when methylating DNAs in vitro. For example, lambda DNA (48,502 bp) contains 3112 CpG sites, and thus a 0.1 mg DNA/ml solution is 19 µM with respect to methyl acceptor sites for the Methyltransferase. This is significant because the recommended concentration of methyl donor, S-adenosylmethionine (SAM), is 160 µM, an 8-fold excess over acceptor sites. Reducing the DNA concentration (< 0.02 mg/ml) gives two advantages. First, the SAM concentration remains high enough to drive the reaction. Second, potential end product inhibition arising from S-adenosyl-L-homocysteine (AdoHcy) generated during the reaction is limited.
    5. Storage of SAM: S-adenosylmethionine is stored at –20°C as 32 mM solution dissolved in sulfuric acid (0.005 M) and 10% ethanol. SAM in this solution stored under ideal conditions remains active for up to 6 months. SAM is unstable at (pH 7.5), 37°C, and should be replenished for reactions incubated longer than 4 hours. Many problems in achieving complete digestion can be alleviated by using fresh SAM.

    References

    1. Renbaum, P. et al. (1990). Nucl. Acids Res. . 18, 1145-1152.
    2. Forney, J.A. and Jack, W.E. (1991). NEB Transcript . 3(1), 5.
    3. Matsuo, K. et al. (1994). Nucl. Acids Res.. 22, 5354-5359.
    4. Doerfler, W. (1983). Ann. Rev. Biochem. . 52, 93-124.
    5. Gruenbaum, Y. et al. (1982). Nature. 295
    6. Bestor, T.H. and Ingram, V.M. (1983). Proc. Natl. Acad. SCI. USA. 80, 5559-5563.
    7. Zacharias, W. et al. (1988). Biochemistry. 27, 2970-2978.
    8. Murchie, A.I. and Lilley, D.M. (1989). J. Mol.Biol. . 205, 593-602.
    9. Ohmori, H. et al. (1978). Nucl. Acids Res. 5, 1479-1485.
    10. Wu, J.C. and Santi, D.V. (1987). J.Biol. Chem. 262, 4778-4786.
    11. Nur, I. et al. (1985). J. Bacteriol.. 164

    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.

    Datacards

    The Product Summary Sheet, or Data Card, 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 the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Is S-adenosylmethionine (SAM) supplied with the Methyltransferase?
    2. What should be considered if the methylation using SssI Methyltransferase is not going to completion?
    3. What is the activity of SssI Methyltransferase in other NEBuffers?
    4. Can SssI Methyltransferase single stranded DNA?
    5. Can SssI Methyltransferase single stranded DNA?
    6. Can SssI Methyltransferase be used for generating a positive control for methylation-specific PCR or bisulfate sequencing?
    7. What is the specific activity of SssI Methyltransferase?
    8. Does SssI Methyltransferase require magnesium in the buffer?
    9. Can DNA be radiolabeled with SssI Methyltransferase?
    10. Can SssI methylated DNA be used to transform E. coli?
    11. What is the molecular weight of SssI (CpG) Methyltransferase?
    1. Recommended Protocol for Methylation of Genomic DNA
    2. Recommended Protocol for 3H label of DNA
    Always add the SAM to the reaction buffer just before doing the methylation reaction, because the SAM is unstable in an aqueous solution. Keep the SAM frozen at -20C for a longer shelf life, fresh SAM is important for optimal methylation reactions.