Dear members,
you all have helped me along very much so far. I have now encountered yet another challenge: I need to design primers for 15 proteins which cover more than one splice-variant of the gene in question (ideally all splice variants).
As i know so far, Ncbi_Nucleotide lists all known variants and i can pick a primer via the button "pick pimers" for each specific primer. However, findig a primer wich picks up a lot of different splice variants may be just a question of luck and a lot of trial and error in my eyes.
Is there any programm/website or loophole which could help me along?
Again, I will deeply appreciate your answers.
Greetings from Munich
Michelle
Ps: Because of this community I could actually get my project going last week. To all members: THANK YOU SOOO MUCH!
Hi Mitzi
I know Aceview website. In this site you can find different variants and also recognize about tissues and each variants too. Although Ensemble always help. As I know you should design primers according to common exon in all variants.
Primer blast is very valuable for checking the accuracy of primers, but I never use this site for primer design. I know about Primer3plus, and i think, it is good enough for primer design.
I hope it helps
Hi, the "pick primers" option on ncbi is your best option when trying to design primers that will pick all transcript variants. You can try designing primers to produce different product sizes to see which will pick all variants. In my experience, primers that produce larger product sizes will almost always pick all transcript variants.
Have you heard of degenerate PCR? they are very well used even today. Just refer any PCR or best the primer vendors who can advise you for it. Other case, just go to uniprot, align sequences and find out which are the common regions that can used to generate a primer which will fish out all varients for that protein. I had in my phd, cloned all varients of MARK protein using a search primer common to all from a cDNA library.
Thank you so much for all you answers!!
They helped me alon a lot!!!
lots of love
Michelle
I have struggled with this same problem, and I found a way to "cheat" Primer-BLAST to effectively include all major transcript variants. Just select manually the start site of the forward primer (e.g. from 1 to 200) and reverse primer (e.g. from 220 to 600) in a way that the binding sites are included in all the transcript variants. After you press search, Primer-BLAST will then ask you, which of the transcript variants are intended or allowed targets and you just select them all (I usually exclude purely predicted variants though). I have contacted NCBI to ask whether they could make this process more user friendly so that one could tell Primer-BLAST immediately from the beginning that all known transcript variants (perhaps purely predicted ones excluded) are INTENDED targets. Currently, it is possible to choose by ticking a box that other transcripts are ALLOWED targets, but in practice, Primer-BLAST tries to avoid including them as far as possible.
I realize I'm 6 years late, but for the sake of those that will arrive at this post in the future - I found a way to get PrimerBlast to give you a primer pair for all transcript variants of the gene of your interest: first, go to NCBI's "Gene" (www.ncbi.nlm.nih.gov/gene) and search for the gene of interest. Copy the ID number of that gene (e.g. for the gene Runx1t1 the ID is 12395). Now go to the PrimerBlast Page and, at the top of the page, choose the tab called "Primers common for a group of sequences". Paste the Gene ID from before in the "PCR Template" field, choose the conditions you're interested in and run the analysis. You should now get primers that cover all (known/predicted) transcript variants of your gene of interest :)
Yes! This new feature "primers common for a group of sequences" is more than welcome!
https://www.biostars.org/p/463987/
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