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McrBC |
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- Isolated from a recombinant source.
- Supplied with 10X Reaction Buffer, GTP, 10 μg control DNA and 100X BSA.
Recognition Site:
Description:
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.
Source:
The two component proteins are purified separately from E. coli K-12 strains containing plasmids encoding McrB and McrC (1).
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).
Reagents Supplied:
NEBuffer 2 (10X)
BSA (100X)
Control Plasmid DNA
GTP (100 mM) (100X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 50% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 10% | | NEBuffer 4: | | 75% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 1.00 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 2
Supplemented with 100 μg/ml Bovine Serum Albumin and 1 mM Guanosine triphosphate
Incubate at
37°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to cleave 1 µg of a plasmid containing a single McrBC site 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.
Concentration:
10,000 units/ml
Unit Assay Substrate:
plasmid containing a single McrBC site
Storage Conditions:
10 mM Tris-HCl
500 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
General 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.
FAQs
- Does McrBC cut hemi-methylated DNA?
- Does McrBC produce blunt or sticky ends?
- Why does my McrBC cleaved DNA smear when run on an agarose gel?
- How much enzyme should be used for cleaving genomic DNA?
- Is extended digestion of McrBC recommended?
- Are there any published papers in which McrBC has been used?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
McrBC is assayed for contaminating endonuclease and exonucleases.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA (HindIII digest)
and 50 units of McrBC incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of McrBC with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of McrBC with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
References
- Sutherland, E. et al. (1992) J. Mol. Biol., 225, 327-334.
- Gowher, H. et al. (2000) EMBO J., 19, 6918-6923.
- Zhou, Y. et al. (2002) Genome, 45, 91-99.
- Stewart, F.J. and Raleigh, E.A. (1998) Biol. Chem., 379, 611-616.
- Panne, D. et al. (1999) J. Mol. Biol., 290, 49-60.
- Stewart, F.J. et al. (2000) J. Mol. Biol., 298, 611-622.
- Raleigh, E.A. (1992) Mol. Microbiol., 6, 1079-1086.
- 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.
Reagents Sold Separately
NEBuffer 2
BSA
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,405,760
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