Hello everyone,
I am designing some primers with Primer-BLAST and this problem keeps happening to me:
I specify the characteristics of the primers, the amplicon, the number of pairs, the organisms in which I want it to test for specificity...
But I am not able to prevent that those pairs with more than one unintended templates from appearing.
I say "more than one" because the first one is always the correct one (I don't know why it appears there but doesn't bother me). Then I have to filter "manually" the pairs that have only this one, (as the one in the attached image), and then choose between them. The real problem is that these pairs could be, maybe one or two in fifty (if there is any).
So, how can I prevent pairs with those "Products on potentially unintended templates" from appearing? Is there any option I am missing?
Thank you!
Dear Dr. Oscar
When I just gave a random human gene accession ID and designed primers for 50 returns (50 pairs result), before the result appeared, I got a page which said that there are some matches with another gene in the database.
.. Finding primers specific to your PCR template (using Primer3 and BLAST)
Input PCR templateNM_000184.3 Homo sapiens hemoglobin subunit gamma 2 (HBG2), mRNA Range1 - 586
Your PCR template is highly similar to the following sequence(s) from the search database. To increase the chance of finding specific primers, please review the list below and select all sequences (within the given sequence ranges) that are intended or allowed targets...
If I tick mark the extra similar gene, I got two primer pair results, each for the two genes.
If I did not tick mark the extra suggested gene and continued, I got 50 pairs result. Except the first one which was my target gene, other appeared as:
Products on potentially unintended templates
> NM_000559.3 Homo sapiens hemoglobin subunit gamma 1 (HBG1), mRNA
I later found that, if you uncheck
... Primer Pair Specificity Checking Parameters
Specificity check
Enable search for primer pairs specific to the intended PCR template ......
in the first page where we enter all the required parameters for primer design, the results are specific to the gene of interest only.
Thank you very much Divya, I worked perfectly.
The problem was, in part, that I had been doing it for some time without the guide activated, and I didn't even see the "extra similar gene" option.
Turn off the "primer pair specificity check". It will stop looking for matches to other genomes and will run faster.
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