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FAQs for CpG Methyltransferase (M.SssI) Protocols for CpG Methyltransferase (M.SssI) FAQs for DNA Modifying Enzymes and Cloning Technical Reference for DNA Modifying Enzymes and Cloning Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE NEBuffer 2 S-adenosylmethionine (SAM)

Home > Products > DNA Modifying Enzymes and Cloning > Methyltransferases > CpG Methyltransferase (M.SssI) CpG Methyltransferase (M.SssI) Catalog # Size Concentration Price Qty   M0226L 500 units 4,000 units/ml $244.00 M0226M 500 units 20,000 units/ml $244.00 M0226S 100 units 4,000 units/ml $61.00 Prices are in US dollars and valid only for US orders. Download: MSDS PDF Methylation Site: Description: The CpG Methyltransferase, M.SssI, methylates all cytosine residues (C5) within the double-stranded dinucleotide recognition sequence 5'...CG...3' (1). 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). 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 Reagents Supplied: NEBuffer 2 (10X) S-adenosylmethionine (SAM) Enzyme Properties Heat Inactivation: 65°C for 20 minutes Reaction & Storage Conditions Reaction Conditions: 1X NEBuffer 2 Supplemented with 160 μM S-adenosylmethionine (SAM) Incubate at 37°C. 1X NEBuffer 2: 10 mM Tris-HCl 50 mM NaCl 10 mM MgCl2 1 mM Dithiothreitol pH 7.9 @ 25°C 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. Protection Assay Conditions: M.SssI is incubated with 1 µg λ DNA in 20 µl 1X NEBuffer 2 [50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl2, 1 mM dithiothreitol], 160 µM S-adenosylmethionine, for one hour at 37°C. The extent of protection by M.SssI is determined by the addition of 30 µl NEBuffer 2 containing 10 units of BstUI restriction endonuclease. Incubation for 1 hour at 60°C is followed by analysis on an agarose gel. Concentration: 4,000 units/ml and 20,000 units/ml Storage Conditions: 10 mM Tris-HCl 1 mM Dithiothreitol 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C Storage Temperature: -20°C Notes General notes: 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. 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. Methylation can be optimized by using fresh SAM. Usage notes: 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). 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. FAQs Is S-adenosylmethionine (SAM) supplied with the Methyltransferase? What should be considered if the methylation using SssI Methyltransferase is not going to completion? What is the activity of SssI Methyltransferase in other NEBuffers? Can SssI Methyltransferase single stranded DNA? Will all the sites in the DNA become methylated by SssI Methyltransferase? Can SssI Methyltransferase be used for generating a positive control for methylation-specific PCR or bisulfate sequencing? What is the specific activity of SssI Methyltransferase? Does SssI Methyltransferase require magnesium in the buffer? Can DNA be radiolabeled with SssI Methyltransferase? Can SssI methylated DNA be used to transform E. coli? What is the molecular weight of SssI (CpG) Methyltransferase? Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product. Quality Assurance Statement: Purified free of contaminating endonucleases and exonucleases. Endonuclease Activity: Incubation of a 50 μl reaction containing 60 units of CpG Methyltransferase (M.SssI) with 1 μg of ΦX174 RF I DNA for 4 hours at 37ºC resulted in < 5% conversion to RFII as determined by agarose gel electrophoresis. 16-Hour Incubation:: Incubation of 100 units of CpG Methyltransferase (M.SssI ) with 1 μg λ DNA in 50 μl of 1X NEBuffer 2 for 16 hours at 37°C resulted in no detectable endonuclease contamination. Exonuclease Activity:: Incubation of 100 units of CpG Methyltransferase (M.SssI) with 1 μg sonicated 3H DNA (105 cpm/μg) for 4 hours at 37°C in 50 μl NEBuffer 2 released < 0.10% of the total radioactivity. References Nur, I. et al. (1985) J. Bacteriol., 164, 19-24. Ohmori, H. et al. (1978) Nucl. Acids Res., 5, 1479-1485. Wu, J.C. and Santi, D.V. (1987) J. Biol. Chem., 262, 4778-4786. Renbaum, P. et al. (1990) Nucl. Acids Res., 18, 1145-1152. 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, 620-621. 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. Reagents Sold Separately NEBuffer 2 S-adenosylmethionine (SAM) Legal Patents: Yissum Research University: U.S. Patent No. 5,296,371