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McrBC > FAQ |  | McrBC FAQ
See the Epigenetics FAQ also.
Q1: Does McrBC cut hemi-methylated DNA?
Q2: Does McrBC produce blunt or sticky ends?
Q3: Why does my McrBC cleaved DNA smear when run on an agarose gel?
Q4: How much enzyme should be used for cleaving genomic DNA?
Q5: Is extended digestion of McrBC recommended?
Q6: Are there any published papers in which McrBC has been used?
Q1: Does McrBC cut hemi-methylated DNA?
A1: Yes. McrBC will cut the DNA (both strands) even if the methylated Cs are on only one strand. There must be at least one methylated C in each half-site for McrBC to cut, but the methylated Cs can be on the same or opposite strands. Therefore, if the DNA is methylated on only one strand, it should still be cut by McrBC.
Q2: Does McrBC produce blunt or sticky ends?
A2: Unfortunately we don't know exactly what ends McrBC leaves. There may even be a mixture of types of ends produced. If McrBC digestion products are to be ligated, we suggest suggest treating the McrBC-digested DNA with Klenow Fragment (3´→5´ exo-) (NEB #M0212) or T4 DNA polymerase (NEB #M0203). This enzyme will remove 3' overhangs and fill-in 5' overhangs, while leaving blunt ends alone. This should leave you with only blunt ends, which could then be ligated.
Q3: Why does my McrBC cleaved DNA smear when run on an agarose gel?
A3: 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 (1). Therefore, 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, producing a smeared rather than a sharp banding pattern.
(1) Stewart, F. J. and Raleigh E. A. (1998) Biol. Chem. 379, 611-616.
Q4: How much enzyme should be used for cleaving genomic DNA?
A4: A 5 to 10-fold excess of enzyme is recommended for cleavage of genomic DNA.
Q5: Is extended digestion of McrBC recommended?
A5: Extended digestion is not beneficial because the enzyme is not stable in reaction.
Q6: Are there any published papers in which McrBC has been used?
A6: Yes:
Holemon, H. et al. (2007) “MethylScreen: DNA methylation density monitoring using quantitative PCR”. Biotechniques, 43, 683-93.
Ordway, J. M. et al. (2007) “Identification of novel high-frequency DNA methylation changes in breast cancer”. PLoS ONE, 2, e1314.
Vaughn, M. W. et al. (2007) “Epigenetic Natural Variation in Arabidopsis thaliana”. PLoS Biol. 5, e174.
Ordway, J. M. et al. (2006) “Comprehensive DNA methylation profiling in a human cancer genome identifies novel epigenetic targets”. Carcinogenesis, 27, 2409-23.
Rangwala, S. H. et al. (2006) “Meiotically stable natural epialleles of Sadhu, a novel Arabidopsis retroposon”. PLoS Genet. 2, e36.
Schumacher, A. et al. (2006) “Microarray-based DNA methylation profiling: technology and applications”. Nucleic Acids Res. 34, 528-42.
Smith, M. J., and Jeddeloh, J. A. (2005) “DNA methylation in lysogens of pathogenic Burkholderia spp. requires prophage induction and is restricted to excised phage DNA”. J. Bacteriol. 187, 1196-200.
Yamada, Y. H. et al. (2004) “A comprehensive analysis of allelic methylation status of CpG islands on human chromosome 21q”. Genome Res. 14, 247-66.
Rabinowicz, P. D. et al. (2003) “Genes and transposons are differentially methylated in plants, but not in mammals”. Genome Res. 13, 2658-64.
Pietrobono et al. (2002). "Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine". NAR, 30, 3278-3285.
Rosado Fantappie et al. (2001). "Lack of DNA Methylation in Schistosoma mansoni". Exp Parasitol. 98, 162-166. [Note: Incorrect source of McrBC stated in this paper. McrBC is available ONLY from NEB.]
Gowher et al. (2000). "DNA of Drosophila melanogaster contains 5-methylcytosine". EMBO 19, 6918 - 6923.
Lyko et al. (2000). "Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila". Nature Genetics 23, 363 - 366.
Pradhan, S. and Roberts, R. J. (2000) “Hybrid mouse-prokaryotic DNA (cytosine-5) methyltransferases retain the specificity of the parental C-terminal domain”. EMBO J. 19, 2103-14.
Santoso et al. (2000). "Control of organ-specific demethylation by an element of the T-cell receptor-alpha locus control region". J.Biol. Chem. 275, 1952-1958.
Burman et al. (1999). "Hypomethylation of an expanded FMR1 allele is not associated with a global DNA methylation deficit". Am. J. Hum. Genet. 65, 1375-1386.
Chotai and Payne (1998). "A rapid, PCR-based test for differential molecular diagnosis of Prader-Willi and Angelman syndromes:. J. Med. Genet. 35, 472-475.
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