Effect of Site-Specific Methylation on Promega Restriction Enzymes
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If little or no cutting is seen with a restriction enzyme, one possibility is that DNA methylation (or lack of methylation, in the case of DpnI) is a problem. The sensitivity of Promega's restriction enzymes to DNA methylation is summarized in this reference table. If the enzyme used is sensitive to methylation, check the genetic characteristics of the bacterial strain or expression system from which the DNA was purified. The interfering type of methylation may be present.
Prokaryotic Methylation
- dcm Cytosine methylase - Methylates the C5 position of the internal cytosine residue in the sequence 5´...CCTGG...3´ and 5´...CCAGG...3´
- dam Adenine methylase - Methylates the N6 position of the adenine residue in the sequence 5´...GATC...3´
Eukaryotic Methylation
- CpG Methylates the C5 position of the cytosine residue in the dinucleotide recognition sequence 5´...CG...3´
- CpNpGp Methylates the C5 position of the cytosine residue in the trinucleotide 5´...CNG...3´ (N = any base)
| Enzyme | Recognition Sequence | dam | dcm | CpG | CpNpG |
|---|---|---|---|---|---|
| ApaI | GGGCCC | i | s(ol) | s(ol) | i |
| BamHI | GGATCC | i | i | i | s(ol) |
| BclI | TGATCA | s | i | i | i |
| BglI | GCCNNNNNGGC | i | i | s(ol) | s(ol) |
| BglII | AGATCT | i | i | i | s(ol) |
| CfoI | GCGC | i | i | s | s(ol) |
| ClaI | ATCGAT | s(ol) | i | s | i |
| DdeI | CTNAG | i | i | i | s(ol) |
| EcoRI | GAATTC | i | i | s(ol) | i |
| HaeIII | GGCC | i | i | i | s(ol) |
| HhaI | GCGC | i | i | s | s(ol) |
| HindIII | AAGCTT | i | i | i | i |
| HpaII | CCGG | i | i | s | s |
| KpnI | GGTACC | i | i | i | i |
| MboI | GATC | s | i | i | i |
| MluI | ACGCGT | i | i | s | i |
| MspI | CCGG | i | i | s | s |
| NheI | GCTAGC | i | i | s(ol) | s(ol) |
| NotI | GCGGCCGC | i | i | s | s |
| PstI | CTGCAG | i | i | i | s |
| PvuI | CGATCG | i | i | s | s(ol) |
| SacI | GAGCTC | i | i | i | n/a |
| SacII | CCGCGG | i | i | s | s |
| SalI | GTCGAC | i | i | s | i |
| ScaI | AGTACT | i | i | i | i |
| SgfI | GCGATCGC | i | n/a | s | n/a |
| SmaI | CCCGGG | i | i | s | s |
| SphI | GCATGC | i | i | i | i |
| TaqI | TCGA | s(ol) | i | i | i |
| XbaI | TCTAGA | s(ol) | i | i | i |
| XhoI | CTCGAG | i | i | s | i |
Key:
s = sensitive to this methylation
i = insensitive to this methylation
s(ol) = overlapping - (sensitive when restriction site overlaps methylation sequence)
n/a = information not available