Product Class: Other

McrBC
NEB2 cloned at NEB recombinant dil_B 37 65 Heat ralbumin Epi

  • Catalog # M0272 was discontinued on September 15, 2023

Product Introduction

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

 

Bioz Badge Exists : True
Catalog # Size Concentration

Product Information

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 (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).
This product is related to the following categories:

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 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

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).

Product 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. 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.

Protocols, Manuals & Usage

Protocols

  1. Protocol for cleavage with McrBC (M0272)

Usage & Guidelines

Tools & Resources

Selection Charts

FAQs & Troubleshooting

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?

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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