I might be going the wrong way about this, but is there a way to check if two or more sequences are present on the same gene?
The reason I want to do it: I've designed primers and taqman probe and want to check for possible cross - amplification with other species.
What it would like in blast: {upstream primer sequence}-{probe sequence}-{downstream primer sequence}.
I can obviously blast each primer + probe and check for % identity - 3' for primers etc.
I tried using :"-" to blast discontinuous sequence but i get a "No significant similarity found" error.
I was wondering if there is something i'm missing.
Thanks.
Hi Stavros Palavouzis ,
To check the specificity of your PCR set, you can just blast your target sequence (the expected product sequence within the gene served as a template for your PCR set) and look if it includes the wanted spectrum (strains-variants-breeds) and if it excludes any other unwanted related species, then you upload your set of sequences and proceed to the phylogenetic analysis (alignments, distances, mismatching...) using any appropriate software (MEGA, BioEdit..).To find repetitive sequences within the same gene across several sequences, you can also use BioEdit.
https://bioedit.software.informer.com/7.2/
https://www.megasoftware.net/
Although they do a pretty good job of keeping it hidden from obvious view, the NCBI primer designing tool, Primer Blast, would probably serve your purpose: https://www.ncbi.nlm.nih.gov/tools/primer-blast/
Enter your target sequence and the primers you have designed (it can also choose primers for you.). Then fill out the "Primer Pair Specificity Checking Parameters" section toward the bottom with your desired choices and Primer Blast will provide info on any possible cross-reactivity. I have not used this in a while, but I seem to remember that much of the possible "cross-reactivity" that it finds would probably never interfere with a real-world PCR--so unless you find some really close similarities, you should be good.
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