McrBC

Catalog #	Concentration	Size	List Price	Your Price
M0272L	10000 units/ml	2500 units
M0272S	10000 units/ml	500 units

Catalog #	Size	List Price	Your Price
M0272L	2500 units
M0272S	500 units

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).

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 Pu^mCG 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 (Pu^mC) 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 Pu^mC elements are as far as 3 kb apart.
Enrichment for undermethylated DNA (11).

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.

Protocols

Protocol for cleavage with McrBC (M0272)

Usage Guidelines

Activity of DNA Modifying Enzymes in rCutSmart™ Buffer

Selection Charts

FAQs

Citations

See more details on Bioz

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]

M0272S_L_v1

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

McrBC

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