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  • McrBC

    Description

    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 or N4-methylcytosine (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).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 2-2010X
    Plasmid DNA for McrBC Linearized methylated (20µl)100 μg/ml
    GTP100X
    BSA-20100X

    Advantages and Features

    Applications

    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 and 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 200 μg/ml BSA and 1 mM GTP
    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

    Storage Temperature

    -20°C

    Storage Conditions

    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

    Quality Control

    Quality Assurance Statement

    • McrBC is assayed for contaminating endonuclease and exonucleases.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

    Notes

    1. 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.
    2. 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.
    3. GTP is more labile than other nucleotides. We recommend aliquoting the 100 mM solution supplied and thawing and diluting as necessary.

    References

    1. Sutherland, E. et al. (1992). J. Mol. Biol.. 225, 327-334.
    2. Gowher, H. et al. (2000). EMBO J.. 19, 6918-6923.
    3. Zhou, Y. et al. (2002). Genome. 45, 91-99.
    4. Irizarry, R.A. et al. (2008). Genome Res.. 18, 780-790.
    5. Hublarova, P. et al. (2009). Int J Gynecol Cancer. 19, 321-325.
    6. Stewart, F.J. and Raleigh, E.A. (2009). Biol. Chem.. 379, 611-616.
    7. Panne, D. et al. (1999). J. Mol. Biol.. 290, 49-60.
    8. Stewart, F.J. et al. (1999). J. Mol. Biol.. 298, 611-622.
    9. Raleigh, E.A. (1992). Mol. Microbiol.. 6, 1079-1086.
    10. Chotai, K.A. and Payne, S.J. (1998). J. Med. Genet.. 35, 472-475.
    11. Burman, R.W. et al. (1999). Am. J. Hum. Genet.. 65, 1375-1386.
    12. Santoso, B. et al. (2000). J. Biol. Chem.. 275, 1952-1958.
    13. Lyko, F. et al. (2000). Nat. Genet.. 23, 363-366.

    Supporting Documents

    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]

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Does McrBC cut hemi-methylated DNA?
    2. Does McrBC produce blunt or sticky ends?
    3.  Why does my McrBC cleaved DNA smear when run on an agarose gel?
    4. How much enzyme should be used for cleaving genomic DNA?
    5. Is extended digestion of McrBC recommended?
    6. Are there any published papers in which McrBC has been used?

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