[Bioperl-l] Restriction Enzyme cuts on Circular plasmids

[Gray, John]

After reading some of your comments about how the site recognition is functioning, I am concerned that there may be another problem.  It commonly occurs that restriction enzyme recognition sites will overlap, and I think this may cause your method to miss some sites.  I am wondering whether it may be necessary to separate the process of site mapping and cleavage. 

For example, BssH II cuts at G^CGCGC, and the sequence of GCGCGCGC theoretically has two cut sites within it.  Of course, your algorithm is similar to reality in that once the enzyme cuts the sequence once, it probably won't be able to recognize the other site.  However, in the test tube what you will actually get is a random distribution of cutting at the two sites.  Traditionally (at least in the software I have used), the site mapping algorithms have returned all possible cut sites.

I am thinking the only way around this would be to first map the sites into an array, and then use that array to either calculate fragment sizes or sequences.  With the possibility of overlapping sites in mind, I still can't think of any way to circumvent the problem of the origin on circular sequences without concatenating the sequence to simulate circularity.

John

-----Original Message-----
From: Rob Edwards [mailto:redwards at utmem.edu] 
Sent: Thursday, October 30, 2003 7:53 PM
To: bioperl-l at portal.open-bio.org
Subject: Re: [Bioperl-l] Restriction Enzyme cuts on Circular plasmids

The following is a quick patch for Bio/Restriction/Analysis.pm so that 
it handles circular sequences correctly if there is another cut site in 
the region that has been linearized. At the moment it won't handle a 
single cut site at that point (e.g. pBR322 has a single EcoRI site at 
the point it is circularized). I am not sure how to deal with this and 
need to think about it (the fragments are right but the cut sites are 
not).

Can someone submit it for me?

I have submitted a Bugzilla report as #1548

120c120,121
< for further analysis. However, this will change the start of the
---
 > for further analysis. This fragment will also be checked for cuts
 > by the enzyme(s). However, this will change the start of the
737c738,749
<                 unshift (@re_frags, $last.$first);
---
 >               my $newfrag=$last.$first;
 >               my @cuts = split /($beforeseq)($afterseq)/i, $newfrag;
 >               my @newfrags;
 >               if ($#cuts) {
 >                # there is another cut
 >                for (my $i=0; $i<=$#cuts; $i+=2) {push (@newfrags, 
$cuts[$i].$cuts[$i+1])}
 >               }
 >               else {
 >                # there isn't another cut
 >                push (@newfrags, $newfrag);
 >               }
 >               push @re_frags, @newfrags;


Thanks

Rob

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