UGENE Forum - Tool for introducing point mutations with PCR

Jan 6^th, 2011 at 11:36pm

silkmoon Offline
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Hi!

I have problem with finding any program dedicated to constructing primers for PCR in purpose of introducing point mutations (primer is mismatched to template in particular position). The tricky thing is calculating Tm (there are many algorithms for it). The only program that can help me to fulfill the task is MELTING (GPL licence) but that program only calculates Tm and isn't created for constructing primers to PCR. It would be nice, if Yours UGENE would be able to help conducting that kind of PCR.

More over it would be easier to conducting in silico PCR in UGENE, if the procedure was similar to that used in PerlPrimer (the GUI is much clearer).

I would like to thank you for such a great program

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Reply #1 - Jan 7^th, 2011 at 1:11am

Konstantin Okonechnikov Offline
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Posts: 173

Hello!

Thanks for your interest in UGENE project!

Actually we're planning to improve PCR analysis capabilities in the next release: add feature to create DNA fragments for cloning "in silico" using PCR, improve visualization for primers and etc. We will consider PerlPrimer as a good example. Point mutations sounds like an interesting new feature, however some clarification is required.

Do you want to be able to locate (construct) all possible "template-matching" primers along with those that may include mismatches OR do you want to be able to modify already located primers and recalculate tm for them?

Concerning the MELTING tool: googled for it but didn't find Sad

Could you please give a direct link?

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Reply #2 - Jan 7^th, 2011 at 3:31am

silkmoon Offline
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Konstantin Okonechnikov wrote on Jan 7^th, 2011 at 1:11am:

Do you want to be able to locate (construct) all possible "template-matching" primers along with those that may include mismatches

Do you mean degenarative PCR (or whatever it is known Wink

)? No. I think there are some tools for it (but I don't know much about that method, so I can't tell for sure).

Konstantin Okonechnikov wrote on Jan 7^th, 2011 at 1:11am:

do you want to be able to modify already located primers and recalculate tm for them?

Yes - exactly, because user must choose on his/her own where position the mismatch (it affects subsequent mutation), so the sequence of the 'preliminary' primer must be known. It'd be great if program could generate sequence of the PCR product with highlighted/annotated mutation.

Konstantin Okonechnikov wrote on Jan 7^th, 2011 at 1:11am:

Concerning the MELTING tool: googled for it but didn't find Sad

Could you please give a direct link?

http://www.ebi.ac.uk/compneur-srv/melting/

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Reply #3 - Jan 7^th, 2011 at 3:42am

silkmoon Offline
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There is one more thing: is it possible to print in UGENE messages from Primer3 ('output', error messages)? Because when primers can't be generated in UGENE, I cannot deduct why (in my case just simply nothing happens and in Log there's no information why Primer3 failed searching for primers).

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Reply #4 - Jan 7^th, 2011 at 7:55pm

Konstantin Okonechnikov Offline
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Posts: 173

Thanks for clarifications

Unfortunately no diagnostics information is available for Primer3 task currently. Most likely the primers are not found due to some restrictions. For example, melting temperature is higher then maximum allowed tm.

Detailed reports and improved logging have the highest priority in the next development cycle. These features are planned to be finished by the end of next week and will be available via development snapshot. I'll keep this thread updated.

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Reply #5 - Jan 7^th, 2011 at 8:45pm

silkmoon Offline
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Konstantin Okonechnikov wrote on Jan 7^th, 2011 at 7:55pm:

Detailed reports and improved logging have the highest priority in the next development cycle.

It'll be very useful, thank you.

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Reply #6 - Jan 13^th, 2011 at 1:38pm

Konstantin Okonechnikov Offline
Global Moderator

Posts: 173

Silkmoon,
did you check the task report info for Primer search task?

It is availble right after the task is finished - click a popup message in the bottom right part of the screen, (See screenshot 1)

You can browse the existing notification messages anytime by double clicking "Memo" pic (Screenshot 2)

From the report you can infer why the primers weren't detected. For example as you can see on screenshot 3 the statistics shows that the tm value is too high for right primer.

screenshot_1.gif (55 KB | )

screenshot_2.gif (58 KB | )

screenshot_3.gif (44 KB | )

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Reply #7 - Jan 13^th, 2011 at 4:13pm

silkmoon Offline
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I have installed the latest snapshot for Linux and in fact it's working as You described. Exactly what I wanted Wink

Thanks!

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Reply #8 - Feb 5^th, 2011 at 5:35am

Peacemaker Offline
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Posts: 12

Hello,
to return to the original question, PrimerX (http://www.bioinformatics.org/primerx/) is a very good online tool for generating Primers for point mutations. It would be great if this could be implemented into ugene, but I' m not sure about the licence situation of primerX.
Best Regards

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Reply #9 - Mar 9^th, 2011 at 8:02am

jot2014 Offline
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Posts: 6

I second these requests

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