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CpG Methyltransferase (M.SssI)

Catalog # Concentration Size List Price Quantity Your Price
M0226L 4000 units/ml 500 units $487.00
$438.30
M0226M 20000 units/ml 500 units $487.00
$438.30
M0226S 4000 units/ml 100 units $120.00
$108.00
Catalog # Size List Price Your Price
M0226L 500 units $487.00
$438.30
M0226M 500 units $487.00
$438.30
M0226S 100 units $120.00
$108.00
Catalog #
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
May be useful for epigenetics - studying cytosine methylation in higher eukaryotes as its specificity mimics the pattern of modification found in their genomes.
  • Blocking restriction enzyme 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
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:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  CpG Methyltransferase (M.SssI) M0226SVIAL -20 1 x 0.025 ml 4,000 units/ml
  NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
  S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM
  CpG Methyltransferase (M.SssI) M0226LVIAL -20 1 x 0.125 ml 4,000 units/ml
  NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
  S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM
  CpG Methyltransferase (M.SssI) M0226MVIAL -20 1 x 0.025 ml 20,000 units/ml
  NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
  S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM
Application Features
  • 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 & 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)

Control Protection Assay

A 20 μl reaction in NEBuffer 2 containing 1 μg of Lambda DNA, supplemented with 160 uM SAM and 1 unit of M.Sss I incubated for 1 hour at 37°C, followed by heat inactivation at 65C for 15 minutes,  results in 95% protection from digestion with 10 units of BstU I in rCutSmart buffer incubated at 60°C for 30 minutes as determined by agarose gel electrophoresis.

Storage Buffer

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

Heat Inactivation

65°C for 20 minutes

Materials Sold Separately
Notes
  • Methylation can be optimized by using fresh SAM.
  • 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.
  • 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.
  • 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.
  • Storage of SAM: S-adenosylmethionine is stored at –20°C as 32 mM solutiondissolved in sulfuric acid (0.005 M) and 10% ethanol. SAM in this solutionstored under ideal conditions remains active for up to 6 months. SAM is unstableat (pH 7.5), 37°C, and should be replenished for reactions incubated longer than4 hours. Many problems in achieving complete digestion can be alleviated byusing fresh SAM.
References
  • Renbaum, P. et al. (1990). Nucl. Acids Res. . 18, 1145-1152.
  • Wu, J.C. and Santi, D.V. (1987). J.Biol. Chem. 262, 4778-4786.
  • Nur, I. et al. (1985). J. Bacteriol.. 164,
  • Forney, J.A. and Jack, W.E. (1991). NEB Transcript . 3(1), 5.
  • Matsuo, K. et al. (1994). Nucl. Acids Res.. 22, 5354-5359.
  • Doerfler, W. (1983). Ann. Rev. Biochem. . 52, 93-124.
  • Gruenbaum, Y. et al. (1982). Nature. 295,
  • Bestor, T.H. and Ingram, V.M. (1983). Proc. Natl. Acad. SCI. USA. 80, 5559-5563.
  • Zacharias, W. et al. (1988). Biochemistry. 27, 2970-2978.
  • Murchie, A.I. and Lilley, D.M. (1989). J. Mol.Biol. . 205, 593-602.
  • Ohmori, H. et al. (1978). Nucl. Acids Res. 5, 1479-1485.
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Specifications
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]
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]
Safety Data Sheets
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