CpG Methyltransferase (M.SssI)

The following reagents are supplied with this product:

NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration
M0226S     -20       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
M0226L     -20       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
M0226M     -20       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

Materials Sold Separately

• NEBuffer 2
• S-adenosylmethionine (SAM)

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.
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.
5. • 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.
6. 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.

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

1. Recommended Protocol for 3H labeling of DNA
2. Recommended Protocol for Methylation of Genomic DNA

• Activity of DNA Modifying Enzymes in rCutSmart™ Buffer
• Effects of CpG Methylation on Restriction Enzyme Cleavage

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, including rCutSmart?
4. Can the SssI Methyltransferase methylate single stranded DNA?
5. Can SssI Methyltransferase be used for generating a positive control for methylation-specific PCR or bisulfate sequencing?
6. What is the specific activity of SssI Methyltransferase?
7. Does SssI Methyltransferase require magnesium in the buffer?
8. Can DNA be radiolabeled with SssI Methyltransferase?
9. Can SssI methylated DNA be used to transform E. coli?
10. What is the molecular weight of SssI (CpG) Methyltransferase?
11. Will any NEB methyltransferases (methylases) work on RNA?

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.

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]

• M0226M_v2
• M0226S_L_v1
• M0226S_L_X_v2

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]

• M0226L_v1_10014347
• M0226S_v1_10014348
• M0226M_v1_10022875
• M0226M_v1_10023317
• M0226S_v1_10024700
• M0226M_v1_10028758
• M0226S_v1_10026143
• M0226L_v1_10030915
• M0226S_v1_10030917
• M0226M_v1_10034569
• M0226L_v1_10034588
• M0226S_v1_10034667
• M0226M_v1_10036699
• M0226M_v1_10036916
• M0226L_v1_10039601
• M0226S_v1_10039594
• M0226L_v1_10043537
• M0226S_v1_10043536
• M0226M_v1_10043567
• M0226L_v1_10045575
• M0226S_v1_10045946
• M0226L_v1_10047067
• M0226S_v1_10048153
• M0226M_v1_10046951
• M0226S_v1_10049707
• M0226L_v1_10049705
• M0226S_v1_10053470
• M0226L_v1_10055040
• M0226M_v1_10053810
• M0226M_v1_10055039
• M0226L_v1_10056363
• M0226S_v1_10055041
• M0226S_v1_10059995
• M0226L_v1_10059998
• M0226M_v1_10062649
• M0226S_v1_10064152
• M0226L_v1_10064783
• M0226S_v1_10064782
• M0226M_v1_10070703
• M0226L_v1_10070701
• M0226S_v1_10070702
• M0226M_v1_10082075
• M0226S_v1_10080091
• M0226S_v1_10082859
• M0226S_v1_10085840
• M0226L_v1_10082858
• M0226L_v1_10086519
• M0226L_v1_10091337
• M0226S_v1_10086520
• M0226L_v1_10094122
• M0226M_v1_10082856
• M0226S_v1_10094123
• M0226L_v1_10100282
• M0226M_v1_10099872
• M0226L_v1_10105430
• M0226S_v1_10106624
• M0226M_v1_10106622
• M0226S_v1_10108746
• M0226L_v1_10108749
• M0226S_v1_10112768
• M0226S_v1_10115299
• M0226L_v1_10115571
• M0226S_v1_10115573
• M0226M_v1_10116037
• M0226L_v1_10121384
• M0226S_v1_10116041
• M0226L_v1_10124258
• M0226M_v1_10126404
• M0226L_v1_10128388
• M0226L_v1_10128960
• M0226M_v1_10128959
• M0226S_v1_10128958
• M0226M_v1_10132852
• M0226L_v1_10133505
• M0226L_v1_10138996
• M0226S_v1_10138995
• M0226M_v1_10140292
• M0226L_v1_10140295
• M0226S_v1_10140289
• M0226L_v1_10144158
• M0226S_v1_10151811
• M0226S_v1_10161319
• M0226M_v1_10151810
• M0226S_v1_10162222
• M0226S_v1_10164648
• M0226L_v1_10162224
• M0226S_v1_10166679
• M0226S_v1_10171073
• M0226L_v1_10173914
• M0226M_v1_10178728
• M0226S_v1_10180288
• M0226L_v1_10180289
• M0226S_v1_10183395
• M0226L_v1_10190579
• M0226L_v1_10191418
• M0226L_v1_10192320
• M0226S_v1_10200649
• M0226M_v1_10201889
• M0226L_v1_10205075
• M0226S_v1_10209015
• M0226M_v1_10209016
• M0226M_v1_10195130
• M0226S_v1_10193026
• M0226S_v1_10210712
• M0226L_v1_10213898
• M0226M_v1_0311803
• M0226S_L_v1_0311802
• M0226S_v1_10218687
• M0226L_v1_10223650
• M0226M_v1_10225765
• M0226S_v1_10231222
• M0226L_v1_10237024
• M0226S_v1_10243048
• M0226L_v1_10245014
• M0226M_v1_10249341
• M0226M_v2_10257138
• M0226L_v2_10256823
• M0226M_v2_10264693
• M0226S_v2_10252416
• M0226S_v2_10263609
• M0226L_v2_10270944
• M0226M_v2_10272266
• M0226L_v2_10276599
• M0226S_v2_10276598
• M0226S_v2_10281694
• M0226L_v2_10282252
• M0226M_v2_10276602
• M0226M_v2_10292559
• M0226S_v2_10290610
• M0226L_v2_10298608
• M0226M_v2_10303905
• M0226M_v2_10310924
• M0226L_v2_10321115
• M0226S_v2_10315685
• M0226M_v2_10321343
• M0226M_v2_10334815
• M0226L_v2_10333006
• M0226S_v2_10333005

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

• CpG Methyltransferase (M.SssI)
• NEBuffer™ 2
• S-adenosylmethionine (SAM)

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