Product Class: Other

CpG Methyltransferase (M.SssI)
NEB2 recombinant 37 65 Heat SAM

M0226CpGMeth_thumb

Product Introduction

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
Catalog # Size Concentration
M0226S 100.0 units 4000 units/ml
M0226L 500.0 units 4000 units/ml
M0226M 500.0 units 20000 units/ml

Product Information

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).
This product is related to the following categories:
Methylation Sensitive and Methylation Dependent Restriction Enzymes for Epigenetics,
Methyltransferases for Epigenetics,
Methyltransferases Products,
This product can be used in the following applications:
Methylated DNA Analysis Products

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

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

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

Product 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.
  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.
  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.

References

  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.

Protocols, Manuals & Usage

Protocols

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

Usage & Guidelines

FAQs & Troubleshooting

FAQs

  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?

Tech Tips

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.

Quality, Safety & Legal

Quality Assurance Statement

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.

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 DataSheets

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

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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