[Bioperl-l] Restriction Enzyme cuts on Circular plasmids

[Rob Edwards]

The best way to do it is to probably cut the sequences, join the first  
and last sequence as is done now, and then see if the enzyme cuts that  
fragment and if so sort out where the fragment should be. I think it is  
reasonably trivial and I can take a look at it soon.

Rob


On Thursday, October 30, 2003, at 08:37  AM, Gray, John wrote:

> Thank you all for the help.
>
> The broken archive search is at  
> http://bioperl.org/pipermail/bioperl-l/.  I will use  
> http://search.open-bio.org in the future.
>
> Cutting circular DNAs is not as simple as concatenating the first and  
> last fragments, as frequently the recognition sequence will span the  
> origin of the plasmid, and in that case the site gets missed.  I have  
> made a work-around within Bio::RestrictionEnzyme by appending the  
> sequence with a short duplication of the beginning:
>
> ## Make circularity function properly AGAIN!!
> ## $concat is set to the length of the recognition sequence -1
> $concat = 5;
> $sequence = uc ($& . (substr $sequence, 0, $concat-1));
>
> The additional sequence must then be later removed from the  
> appropriate fragment.  (Here I am only after the length of the  
> fragments):
>
> ## Cut the sequence with the restriction enzyme object.
> @fragments = eval{ $re->cut_seq($seq); };
> $@ && die "\n*** TROUBLE CUTTING:\n$@\n";
>
> ## pull lengths from array of sequences
> my @fraglen;
> my $i = 0;
> foreach (@fragments) {
>         $fraglen[$i] = length $_;
>         $i++;
>         }
> ## Sum first and last fragment lengths due to circularity, remove last
> $fraglen[0] = $fraglen[0] + $fraglen[@fraglen-1] - $concat;
> pop @fraglen;
>
>
> This works well so far, but it is cumbersome because I must modify the  
> sequence in a specific way for each enzyme recognition site length.
>
> Reading the documentation for the new Bio::Restriction module I see  
> that it has many improvements which would have made my code much  
> easier, but it is not clear that the methods address the problem that  
> I raised above.  Anyone have any idea?
>
> Thanks again-
>
> John Gray
>
>
>
>
> -----Original Message-----
> From: Rob Edwards [mailto:redwards at utmem.edu]
> Sent: Wednesday, October 29, 2003 6:31 PM
> To: Gray, John
> Cc: bioperl-l at portal.open-bio.org
> Subject: Re: [Bioperl-l] Restriction Enzyme cuts on Circular plasmids
>
> The new Bio::Restriction modules should tell you the correct sizes of
> circular plasmids. This will do exactly what you want. The old
> Bio::Tools::RestrictionEnzyme does not have this ability, though you
> can just merge the first and last fragments of a sequence.
>
> The newer Bio::Restriction are (I believe) only available in BioPerl
> 1.3 and later. This is the upcoming release and the first RC seems
> quite stable.
>
> You should probably tell someone about the searches not working, but I
> guess you just have :)
>
> Rob
>
>
>
> On Wednesday, October 29, 2003, at 10:39  AM, Gray, John wrote:
>
>> I am trying to find documentation for the Bio::Restriction module, to
>> see if it can properly predict restriction fragment sizes for circular
>> plasmids.
>>
>>
>>
>> I'm sorry to have to ask.  The tutorial at
>> http://www.bioperl.org/Core/Latest/bptutorial.html says that
>> Bio::Restriction replaces the older Bio::RestrictionEnzyme, which I
>> have used.  I can't find any documentation for this method at
>> http://doc.bioperl.org/releases/bioperl-1.2.3/.
>>
>>
>>
>> The Seq documentation does not provide any description of the
>> 'is_circular' method, and I was hoping that I could set the Seq object
>> to be circular, and then just run something like
>> $enyme_object->cut_seq($seq_object) to get my list of strings
>> containing the resulting fragments.
>>
>>
>>
>> Thank you for your help.
>>
>>
>>
>> PS.  When I try and search the Bioperl-l archive, it doesn't return
>> any matches, even when I try to match the word 'bioperl'.  Should I
>> tell someone about this?
>>
>>
>>
>> ---------------------------------------------------------------------- 
>> -
>> --------------
>> John T. Gray, Ph.D.
>> Director, Vector Development & Production
>> Experimental Hematology Division
>> Hematology-Oncology
>> St. Jude Children's Research Hospital
>> D2038A, Mail Stop 341
>> 332 N. Lauderdale Street
>> Memphis, TN 38105
>>
>> (901) 495-4729 phone
>> (901) 495-2176 fax
>>
>> John.Gray at stjude.org <mailto:John.Gray at stjude.org>
>>
>>
>>
>> _______________________________________________
>> Bioperl-l mailing list
>> Bioperl-l at portal.open-bio.org
>> http://portal.open-bio.org/mailman/listinfo/bioperl-l
>
>
>
>
> _______________________________________________
> Bioperl-l mailing list
> Bioperl-l at portal.open-bio.org
> http://portal.open-bio.org/mailman/listinfo/bioperl-l