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McrBC

Catalog # Concentration Size List Price Quantity Your Price
M0272L 10000 units/ml 2500 units
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M0272S 10000 units/ml 500 units
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Catalog # Size List Price Your Price
M0272L 2500 units
M0272S 500 units
Please Inquire
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

McrBC is an endonuclease which cleaves DNA containing methylcytosine* on one or both strands. Sites on the DNA recognized by McrBC consist of two half-sites of the form (G/A)mC. These half-sites can be separated by up to 3 kb, but the optimal separation is 55-103 base pairs.

*5-methylcytosine or 5-hydroxymethylcytosine

  • McrBC requires GTP for DNA cleavage
  • Supplied with NEBuffer 2, Recombinant Albumin and 100x GTP

 

Recognition Site:

McrBC is an endonuclease which cleaves DNA containing methylcytosine* on one or both strands. McrBC will not act upon unmethylated DNA (1). Sites on the DNA recognized by McrBC consist of two half-sites of the form (G/A)mC. These half-sites can be separated by up to 3 kb, but the optimal separation is 55-103 base pairs (2,3). McrBC requires GTP for cleavage, but in the presence of a non-hydrolyzable analog of GTP, the enzyme will bind to methylated DNA specifically, without cleavage (4).
*5-methylcytosine or 5-hydroxymethylcytosine (5).

Linearized plasmid (methylated or unmethylated), containing one McrBC site, incubated with McrBC. Lane 1, unmethylated DNA; Lane 2, methylated DNA; Lane 3, Marker: Lambda DNA BstEII digest.
Human and Drosophila genomic DNA incubated with McrBC. 60-90% of CpGs in vertebrate DNA are estimated to be methylated (6) while methylated CpGs are extremely rare in Drosophila DNA (7). Lane 1, Human DNA; Lane 2, Human DNA incubated with McrBC; Lane 3, Drosophila DNA; Lane 4, Drosophila DNA incubated with McrBC; Lane 4, Marker: Lambda DNA BstEII digest.
Product Source
The two component proteins are purified separately from E. coli K-12 strains containing plasmids encoding McrB and McrC (1).
Application Features
CpG methylation status:
McrBC is a tool for determining the methylation state of CpG dinucleotides (6-10). McrBC will act upon a pair of PumCG sequence elements, thereby detecting a high proportion of methylated CpGs, but will not recognize HpaII/MspI sites (CCGG) in which the internal cytosine is methylated.
Detection of cytosine-methylated DNA:
The very short half-site consensus sequence (PumC) allows a large proportion of the methylcytosines present to be detected. Even DNA which is not heavily methylated can be detected, as a low level of cleavage occurs even when the PumC elements are as far as 3 kb apart.
Enrichment for undermethylated DNA (11).

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to cleave 0.5 µg of a plasmid containing multiple McrBC sites in 1 hour at 37°C in a total reaction volume of 50 µl. A pilot titration of enzyme is recommended for cleavage of genomic DNA.

Reaction Conditions

1X NEBuffer™ 2
Supplement with 1 mM GTP and 200 µg/ml Recombinant Albumin, Molecular Biology Grade
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)

Activity in NEBuffers

NEBuffer™ 1: 50%
NEBuffer™ 2: 100%
NEBuffer™ 3: 10%
NEBuffer™ 4: 75%

Diluent Compatibility

Storage Buffer

10 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
500 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 minutes

Companion Products
Materials Sold Separately
Notes
  • McrBC makes one cut between each pair of half-sites, cutting close to one half-site or the other, but cleavage positions are distributed over several base pairs approximately 30 base pairs from the methylated base (2). Therefore, the enzyme does not produce defined DNA ends upon cleavage. Also, when multiple McrBC half-sites are present in DNA (as is the case with cytosine-methylated genomic DNA) the flexible nature of the recognition sequence results in an overlap of sites, and so a smeared rather than a sharp banding pattern is produced.
  • McrBC cleavage of the supplied 4.3 kb linear, methylated control plasmid DNA produces several fragments between approximately 700 bp and 2.3 kb in size.
  • GTP is more labile than other nucleotides. We recommend aliquoting the 100 mM solution supplied and thawing and diluting as necessary.
References
  • Sutherland, E. et al. (1992). J. Mol. Biol.. 225, 327-334.
  • Chotai, K.A. and Payne, S.J. (1998). J. Med. Genet.. 35, 472-475.
  • Burman, R.W. et al. (1999). Am. J. Hum. Genet.. 65, 1375-1386.
  • Santoso, B. et al. (2000). J. Biol. Chem.. 275, 1952-1958.
  • Lyko, F. et al. (2000). Nat. Genet.. 23, 363-366.
  • Gowher, H. et al. (2000). EMBO J.. 19, 6918-6923.
  • Zhou, Y. et al. (2002). Genome. 45, 91-99.
  • Irizarry, R.A. et al. (2008). Genome Res.. 18, 780-790.
  • Hublarova, P. et al. (2009). Int J Gynecol Cancer. 19, 321-325.
  • Stewart, F.J. and Raleigh, E.A. (2009). Biol. Chem.. 379, 611-616.
  • Panne, D. et al. (1999). J. Mol. Biol.. 290, 49-60.
  • Stewart, F.J. et al. (1999). J. Mol. Biol.. 298, 611-622.
  • Raleigh, E.A. (1992). Mol. Microbiol.. 6, 1079-1086.
Quality Control Assay
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 Data Sheets
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