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in silico PCR and cloning with Gibson Assembly (Read 14432 times)
Dec 6th, 2012 at 8:27pm

randolf   Offline
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Hi there,

I'm a litte biologist and want to use Ugene for modeling of lab sequence modification experiments. In this context, I have some questions.

Consider the following situation: I want to add restriction enzyme cut sites to a PCR fragment in the lab. For this purpose I have primers with overhangs. Is there a way to easily construct the resulting PCR sequence from template and primers with Ugene.

Another thing I wonder: I have overlapping PCR products (20 nucleotides overlap) and want to assemble them with Gibbson or Yeast assembly. This means they fuse at the overlaps. Can this be easily done with Ugene. I tried around with "DNA assambly" and "Align to reference", but this doesn't seem to work with fasta format and has another pupose anyway.

I have primers with non-annealing overhangs and I want to find the annealing positions on a sequence. At the moment I do this search with Smith-Waterman-algorithm and large gap panalty, but this is far from fast and efficient.

Hints will be greatly appreciated!
Regards,
Thomas
 
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Reply #1 - Dec 7th, 2012 at 6:17am

Konstantin Okonechnikov   Offline
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hi Thomas!
Quote:
Consider the following situation: I want to add restriction enzyme cut sites to a PCR fragment in the lab. For this purpose I have primers with overhangs. Is there a way to easily construct the resulting PCR sequence from template and primers with Ugene.

The PCR design feature request, which is similar to what you've written, is currently under active development and will be available soon.

Meanwhile you can do the following:

1) Use Analyze->Primer 3... dialog to search for particular primers in your sequence by filling them into fields below Pick left/right primer boxes.  After primer pair is found, one can use menu item Cloning->Make PCR product to create corresponding fragment, or just select product region (hold Shift key when selecting primer annotations) and export the selected sequence.

2)  If you would like to add primers as blunt overhangs to your sequence fragment you can use menu Edit sequence->Insert subsequence to add the overhangs.

Quote:
Another thing I wonder: I have overlapping PCR products (20 nucleotides overlap) and want to assemble them with Gibbson or Yeast assembly. This means they fuse at the overlaps. Can this be easily done with Ugene. I tried around with "DNA assambly" and "Align to reference", but this doesn't seem to work with fasta format and has another pupose anyway.

Could you please describe in more detail, what would you like to achieve?  I am not sure, if I understand your intentions correctly.

DNA Assembly in UGENE refers to assembly of contig sequences from overlapping sequence fragments. Alignment to reference is alignment of short sequence fragments to reference sequence. This methods are mostly used in context of sequencing.

Quote:
I have primers with non-annealing overhangs and I want to find the annealing positions on a sequence. At the moment I do this search with Smith-Waterman-algorithm and large gap panalty, but this is far from fast and efficient.


How big is a list of your primers? I think this task can be performed with primer3.
 
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Reply #2 - Aug 14th, 2013 at 11:12pm

vixen sjlin   Offline
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Many free programs do in-silico PCR, however, very few of them can perform primers with 5' overhangs, which is the regions not aligned to the template.  I do recommended Ugene to consider to add 5' overhang functions.  Two good in-siligo PCR program with 5' overhang function are jPCR and Serial Cloner
 
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Reply #3 - Aug 18th, 2013 at 5:23am

Ilya Flyamer   Offline
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I've recently asked a question about Gibson assembly in Russian forum and didn't get any reply Sad It would be really helpful to have this feature.
 
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