101 things you (maybe) didn’t know about MacVector: #28 – Identifying Methylation Blocked Restriction Sites

A big thanks to Jeffrey Dvorin at Boston Children’s Hospital for this great suggestion.

Most common laboratory strains of E. coli contain a number of methylase enzymes that modify DNA residues, preventing certain restriction enzymes from cutting DNA isolated from those strains. The two most relevant enzymes are the Dam methylase that methylates the A in the sequence GATC and the Dcm methylase that methylates the second C in the sequences CCAGG and CCTGG. For a more detailed description of the Dam and Dcm methylases, check out this page on the New England Biolabs website.

Some enzymes are entirely blocked by this methylation. For example MboI recognizes the sequence GATC, the same as the Dam methylase recognition sequence, but is blocked from cutting if the A is methylated. Thus MboI does not cut DNA isolated from a Dam+ E. coli strain. However, Sau3A also recognizes GATC, but is unaffected by the methylation, so cuts Dam+ E. coli DNA completely.

The situation with other enzymes can be more complicated – ClaI recognizes the sequence ATCGAT, but will not cut if either of the A residues are methylated. That means that if the site is flanked by a 5′ G or a 3′ C residue (e.g. GATCGAT or ATCGATC) then one of the A’s will be methylated and the site will not be cleaved. However, other sites (e.g. TATCGATT or AATCGAT etc) do not contain Dam methylase sites and so will be cleaved normally.

MacVector does not have any direct support for identifying cleavage sites that are blocked by the Dam or Dcm methylases. However, thanks to a trick that Jeff Dvorin suggested, there is an easy way to display them on maps. The basic idea is that you modify the restriction enzyme files you normally use for RE searching to include customized methylation site entries. Lets look at an example using the ClaI enzyme discussed above;


In the screenshot above, I’ve modified the default Common Enzymes.renz restriction enzyme file to include a new site called DAM-ClaI that is almost identical to the normal ClaI site except that it contains an additional 3′ C residue. Because DNA is double-stranded and MacVector searches both strands, this site will find both GATCGAT and ATCGATC. So now, if I search a DNA sequence with both of these enzymes selected, I can easily see which sites will be blocked by Dam methylase activity. Here’s an example – its obviously artificial as you would never normally see so many ClaI sites in such a short piece of DNA – but you can clearly see the ClaI sites that would be blocked as they have both ClaI and DAM-ClaI sites at the same position on the DNA strand;


Note that the two DAM-ClaI sites are ...GATCGAT... and ...ATCGATC... demonstrating that the single DAM-ClaI site in the .renz file does indeed identify both types of site.

So, we have a simple way of identifying sites that will be blocked by the Dam and Dcm methylases. All you have to do is individually enter the sites listed on the New England Biolabs page into each of your restriction enzyme files and and you are done! What? Think that’s too much work? Of course we have a file to help you out! If you download this file from our website, we’ve already included all the methylation-sensitive restriction enzyme sites and labelled them based on their sensitivity to either the Dam or Dcm methylase. You do need to add all of the sites to your favorite enzyme files, but that is pretty simple;

(a) Use File | Open and locate and open the Methylation-Sensitive Restriction Enzymes.renz file.

(b) Use File | Open and locate and open your favorite restriction enzyme file (e.g. Common Enzymes.renz or New England Biolabs.renz).

(c) Bring Methylation-Sensitive Restriction Enzymes.renz to the front, choose Edit | Select All, followed by Edit | Copy.

(d) Switch to your target .renz file and choose Edit | Paste. The DAM and DCM enzymes will get pasted into the file. Save the file.

Now, whenever you use that file for searches, the methylation sensitive versions will show up in the results. However, note the following caveats;

(i) If you run a search using “Selected Enzymes” (and that is the default for the automatic search that is run whenever you view the Map tab of an open DNA sequence), you must also make sure any corresponding DAM- or DCM- enzymes are checked to be sure they will appear in the results.

(ii) If you limit the results of searches based on the number of cut sites, you may not see the results you expect. For example, in the above ClaI example there were 4 ClaI sites and 2 DAM-ClaI sites. If you set the filter to only show enzymes that cut twice or less you would see only the DAM-ClaI sites. Conversely, if you set the filter to show enzymes that cut between 3 and 5 times, you would see the ClaI sites, but not the DAM-ClaI sites.

We’ll likely incorporate this functionality into a future version of MacVector with a simplified user interface. But for now, this is a great way to identify those pesky methylation blocked sites and works for any version of MacVector.

This is an article in a long running series of tips to help you get the most out of MacVector. If you want to get notified every time a new tip gets published, follow us @MacVector on twitter (or check the feed for the hashtag #101MacVectorTips) or like us on Facebook.

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