McrBC |NEB酶试剂 New England Biolabs

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产品信息

Recognition Site:
McrBC |

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).

McrBC |
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.
McrBC |
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.

产品来源

The two component proteins are purified separately from E. coli K-12 strains containing plasmids encoding McrB and McrC (1).

产品类别:
Discontinued (<3 years)

  • 特性和用法

    单位定义

    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.

    反应条件

    1X NEBuffer™ 2
    Supplement with 1 mM GTP 和 200 µg/ml 重组白蛋白，分子生物学级
    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)

    在不同缓冲液中的活性

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

    稀释兼容性

    • 稀释液 B

    贮存溶液

    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

    热失活

    65°C for 20 minutes

  • 优势和特性

    应用特性

    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).

  • 相关产品

    相关产品

    • t1010-monarch-plasmid-miniprep-kit
    • Monarch® DNA 胶回收试剂盒
    • Monarch® PCR & DNA 纯化试剂盒（5 μg）

    单独销售的组分

    • NEBuffer 2
    • 重组白蛋白, 分子生物学级（不含动物成分）

  • 注意事项

    1. McrBC 在每对半位点之间切割一次，切割点靠近某一个半位点，但是通常距最近的甲基化碱基大约 30 bp（2）。因此，此酶的切割不会产生确定的 DNA 末端。除此之外，当 DNA 上存在多个 McrBC 半位点时（例如胞嘧啶甲基化修饰的基因组 DNA），识别序列的可变性会导致位点重叠，从而产生弥散状而不是细窄的切割条带。
    2. McrBC 切割随附的 4.3 kb 线性甲基化对照质粒 DNA 会产生几个大小约为 700 bp 和 2.3 kb 的片段。
    3. GTP 比其它核苷酸更不稳定。建议将随附的 100 mM 溶液进行分装，按需解冻和稀释。

  • 参考文献

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

操作说明、说明书 & 用法

  • 操作说明

    1. Protocol for cleavage with McrBC (M0272)

  • 使用指南

    • Activity of DNA Modifying Enzymes in rCutSmart™ Buffer

工具 & 资源

  • 选择指南

    • DNA Methylation Table
    • Restriction Enzymes for Epigenetics Selection Chart

FAQs & 问题解决指南

  • FAQs

    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?
    7. Will McrBC work in rCutSmart Buffer?