[Bioperl-l] Restriction Enzyme cuts on Circular plasmids

[pblaiklo at 110.net]

   I am rewriting Bio::Restriction::Analysis to fix the circularity bug (and 
others), using similar algorithms to the ones you describe. Right now it 
doesn't handle overlapping sites well, but this should be pretty easy to fix. 
Using an array of cut positions also lets you keep the start and end points 
of each restriction fragment. This will make it easier for users to turn 
restriction fragments into features of the target sequence.
 
Peter Blaiklock

On Friday 31 October 2003 09:36, Gray, John wrote:
> 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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