Dam and Dcm Methylation Restriction Block Checker (pUC19)
Inspect pUC19 restriction sites for cleavage blocks caused by the two E. coli host methyltransferases the engine models, Dam (5′-GATC-3′) and Dcm (5′-CCWGG-3′). CpG methylation is not modelled. The workspace opens an MboI and XbaI digest of pUC19 with the dam+/dcm+ host set: every MboI site stays uncut, so XbaI alone opens the circle into one 2,686 bp fragment. Toggle to unmethylated DNA to see which sites change.
Opens the 2,686 bp pUC19 digest (GenBank L09137.2) with a dam+/dcm+ host. The engine models Dam and Dcm only, against McClelland et al. 1994: on this sequence all 15 MboI (GATC) sites are Dam-methylated and shown as not cut, while the XbaI site (TCTAGA at 423) has no overlapping GATC, is unmethylated and cuts. The outcome is shown per site rather than as a global block.
Check flanking bases for hidden Dam (GATC) and Dcm (CCWGG) boundary overlaps
Standard laboratory E. coli K-12 cloning hosts (including DH5α, TOP10, Mach1, and NovaBlue) express two endogenous housekeeping methyltransferases: Dam methylase, which transfers a methyl group to the N6 position of adenine in 5′-GATC-3′, and Dcm methylase, which methylates the internal cytosine at the C5 position in 5′-CCAGG-3′ and 5′-CCTGG-3′ (5′-CCWGG-3′). Some restriction enzymes—such as DpnI (which requires methylated GATC) or MboI (which is blocked by methylated GATC at every site)—have intrinsic methylation sensitivity built directly into their core motif.
More insidious are conditional boundary overlaps, where only a subset of recognition sites on a plasmid happen to share 1 to 3 terminal bases with an adjacent GATC or CCWGG sequence. For example, BclI (TGATCA) contains GATC internally and is always blocked in dam+ hosts, whereas XbaI (TCTAGA) is blocked only when its flanking bases complete an overlapping GATC (TCTAGATC or GATCTAGA). In pUC19 every MboI site is blocked in a dam+ host, while the single XbaI site at 423 is not.
Match host strain methylation state to your simulated digest settings
When inspecting the pUC19 reference in the plasmid workspace, toggling between a dam+/dcm+ host and unmethylated DNA reveals how methylation state alters effective site availability without changing a single nucleotide of the FASTA or GenBank sequence. If a plasmid carries two sites for an enzyme—one unmethylated and one where a methylated base falls on a blocked position—digesting DNA from DH5α can cut the first while leaving the second intact.
Always record the propagation host strain alongside plasmid miniprep stocks. When designing a new multiple cloning site or synthetic gene cassette, check the few nucleotides immediately upstream and downstream of every planned restriction site so you do not inadvertently create a 5′-GATC-3′ or 5′-CCWGG-3′ overlap that forces a later passage through a dam−/dcm− strain.
Equations and variables
Boundary overlap between recognition motif R and methylase motif M
Overlap(R, M) = (R[L − k : L] == M[0 : k]) or (M[Lm − k : Lm] == R[0 : k])
- R, M
- Recognition sequence of length L and methyltransferase motif of length Lm (e.g. GATC or CCWGG)
- k
- Number of shared boundary nucleotides (1 ≤ k < min(L, Lm)) placing a methylated base inside R
Fragment count when some sites of an enzyme are blocked by methylation
Nfragments = m − nblocked (circular plasmid; one linear molecule when a single site cuts)
- Nfragments
- Fragments from a complete digest of the circular plasmid
- m
- Total occurrences of the recognition sequence on the circular plasmid
- nblocked
- Sites where a host-methylated base falls on a position the enzyme does not cut
Scope and limitations
- A block is reported only where a methylated base falls where McClelland et al. 1994 lists the enzyme as not cutting; sites marked slow, partial or variable are shown as not recorded, never as cutting.
- Plasmid DNA isolated from standard DH5α, TOP10, or XL1-Blue strains is dam+/dcm+, whereas PCR amplicons and DNA propagated in dam−/dcm− strains (such as JM110 or ER2925) are unmethylated at those motifs.
- Dcm methylation in stationary-phase E. coli cultures is occasionally incomplete, producing a confusing mixture of cleaved and uncleaved bands rather than an all-or-none block.
Sources and provenance
- GenBank pUC19 record L09137.2Existing source notice: NCBI public domain
Versioned sequence and SHA-256 are pinned in the shipped example.
- Marinus and Løbner-Olesen 2014 DNA methylation in Escherichia coliArticle citation; Dam and Dcm methyltransferase specificity
- McClelland, Nelson and Raschke 1994: effect of site-specific modification on restriction endonucleasesArticle citation; the methylation-sensitivity table the engine reads
Technical questions
How can an enzyme like XbaI (TCTAGA) be blocked by Dam methylation when GATC is not its recognition sequence?
Dam methylase methylates the adenine in every 5′-GATC-3′ sequence. When an XbaI site is immediately followed by TC (5′-TCTAGATC-3′) or preceded by GA (5′-GATCTAGA-3′), an adenine on the site becomes part of an overlapping GATC and is methylated. The workspace reports the outcome McClelland et al. 1994 records for that enzyme at that methylated position. The XbaI site at 423 in pUC19 has no such overlap, so it is shown as cutting.
Why does a PCR product cut completely with an enzyme that failed to cut the parent plasmid?
Thermostable DNA polymerases incorporate unmodified dATP, dCTP, dGTP, and dTTP during in vitro amplification, erasing all host Dam, Dcm, and CpG methylation marks. A site that was blocked by overlapping methylation in plasmid miniprep DNA from DH5α becomes fully cleavable in the PCR amplicon.
How do you bypass a Dam or Dcm restriction block without changing the plasmid sequence?
Transform the plasmid into a methylation-deficient E. coli host strain such as JM110 or ER2925 (dam−/dcm−) and prepare fresh plasmid DNA, or amplify the backbone by PCR, which leaves the product unmethylated. Toggling the host to unmethylated in the workspace shows the same effect.
Connected workflows
- Virtual restriction digest and fragment sizes
Simulate double-digest fragment lengths under explicit methylation settings.
- GenBank and FASTA plasmid mapping
Inspect the flanking nucleotide sequence around every pUC19 restriction site.
- Primer-pair Tm and template matching
Amplify unmethylated template DNA by PCR when host methylation blocks a site.