I am using BLAST for the first time in order to verify the targeting specificity of primers. I have a primer pair that produce two products from the target gene and a variant of it. Both of these are the same length (144bp) so I think this should be okay and won't affect targeting specificity nor the readings for the melting curve. However, I would like confirmation on this and reading through BLAST tutorial papers has not answered this question for me?
Any thoughts from BLAST pros?
Hi William,
So, if I understand correctly, you have a couple of previously designed primers (i.e. they are not from the BLAST primer tool itself). And when you tested them with the tool, they perfectly matched two similar genes and produced an amplicon of the same length.
In this case, the melting temperature of the two could be identical, but only if the amplicons have exactly the same sequence (I mean between the primers also). Therefore, you could do an alignment with both amplicons to check it. If it is identical there will be no problem.
I hope this was helpful.
I'd recommend designing a new pair of primers. You won't be able to be 100% certain that the primers are equally efficient at amplifying both loci. Even if you were sure, you'd still have to adjust your calculations based on having 4 copies per genome instead of the usual 2 (assuming your organism is diploid).
William MacIntosh-Smith to your question:
"Is this alignment tool a feature in BLAST, or would I need another tool for this?"
I usually use MEGA software to perform the alignments: https://megasoftware.net/
But you can also do it with this BLAST tool: https://blast.ncbi.nlm.nih.gov/BlastAlign.cgi
In this case you just put one sequence on top and the other at the bottom, it's quite simple!
In my previous message I forgot to tell you that: maybe both amplicons could also have the same melting temperature if they share their GC-content, even if they are not a perfect alignment (but I am not entirely sure of this).
There are many tools for primer designing by using insilico, I can suggest more reading pubmed articles. Infact for alignment you can use blast, mega, clustal Omega etc free tools.
https://pubmed.ncbi.nlm.nih.gov/25697657/
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