I want to design universal primers for a protein. In case I am getting the terminology wrong - what I want are primers for RT-qPCR that will amplify the particular protein from multiple species in one go. So far I have been playing around with the following:
Searching for the protein in NCBI under 'nucleotides'; this yields many thousands of results.
Downloading several FASTA sequences and pasting them into the conservative primer generator web application PrimerIdent, which then outputs a list of primers, but the % identity/match is very low.
I have also gone down several other routes with no luck - highly likely a result of my lack of expertise in this area.
Is there a (relatively!) straightforward workflow to follow for this? Given I am not doing this many times, automation via programming would be less desirable than web-based applications (but happy to consider!). I would also be happy to be directed to earlier posts or websites if this has already been covered, but after a day of googling I am feeling relatively stuck!
Please check your terminology: there are no primers for proteins and you can't amplify protein or measure protein expression by PCR. To be precise, what you can attempt is to design universal primers for the coding sequences of genes which have have orthologous proteins as their product. You could download some CDS for closely related organism, do a multiple sequence alignment and inspect the MSA for highly identical stretches of sequence. However, for protein coding genes the nucleotide sequence is not generally highly conserved, unlike for some RNA genes (e.g. bacterial 16s) for which universal primers can be derived. Therefore, for more than a handful of species, or more distantly related species, this approach is not going to work at all, and therefore there will not be a straight forward approach.
I would start by looking at a multiple alignment of the gene between all species of interest, and identify sections of the gene that are identical or near identical in all species, then design my proteins for these regions.
Creating so called universal primers is actually a really hard thing to do, because at the heart of the question lies an oxymoron:
The region of DNA you wish to amplify is highly similar. Similar enough that you call them the same protein.
The rest of the DNA in the organism must, therefore, be very different. Different enough that you call them different species.
Typically we prime in region 2 for two very good reasons:
DNA is so prevalent that designing primers inside a coding region is almost a guarantee for getting non-target genome contamination - Bacterial, Viral, Human, Cat, its all in there. This would also be true for a 'universal primer', and would make any kind of analysis a lot less convincing.
Coding regions tend to be less unique by the nature of copy-paste-mutate evolution, and certainly less complex due to, amongst other things, codon usage bias and amino acid bias. Emily's advice will certainly get you half of the way there for priming non-coding regions - but its the same problem at the end of the day - targeting conserved regions means targeting low-complexity regions due to their non-random nature, and thus primer specificity becomes an issue. If the region has to be conserved across many species, it becomes a BIG issue. Since you can't even check specificity in many organisms since they don't have a fully mapped genome, this is likely to be a constant problem which will make you constantly ask why you didn't just design another primer pair.
EDIT: Just saw that you want these for RT-qPCR. Now it all makes sense :)
The amplification efficiencies of the same primer in a different genome is going to be... well... different. You will have to normalize for house keeping genes for each species, so in addition to all the issues mentioned above, using the same primer is not going to save you any work at all. An analogy would be like testing who's the better snowboarder, you or a 2 year old child, given the same snowboard. That would be totally unethical.
You should give him a tiny snowboard first. Then if he makes it to the bottom of the slope normalise to account for his tiny legs.
I have managed to get Universal Primers for cloning ortholog genes with a variable degree of success that was depending upon the degree of conservation of the studied protein among species (obvious..).
I've been isolating DNA from (unknown) plant, and could clone in one case 9 out o 10 the gene of interest, and in a case of a better conserved gene, the rate of success was 10/10..
I followed the CODEHOP approach with minor modifications
You align your protein sequences and follow the CODEHOP recommendations
The program will provide you with some oligos to try
Thus, I align the corresponding DNA sequences, and try to see how well the primers provided by CODEHOP align to those sequences. Minor changes are done to minimize unmatched bases. I use known >DNA sequences, but the cloning can be done in unknown sequences. The alignment will alert you of the degree of conservation, and thus for the expected rate of success
(Optional) An optimization of the PCR reaction is done following the Taguchi method to figure out the best DNA, primer concentration, Mg++ and Taq polymerase concentrations. A search with Google with "Taguchi primers" will reveal you the details
Please check your terminology: there are no primers for proteins and you can't amplify protein or measure protein expression by PCR. To be precise, what you can attempt is to design universal primers for the coding sequences of genes which have have orthologous proteins as their product. You could download some CDS for closely related organism, do a multiple sequence alignment and inspect the MSA for highly identical stretches of sequence. However, for protein coding genes the nucleotide sequence is not generally highly conserved, unlike for some RNA genes (e.g. bacterial 16s) for which universal primers can be derived. Therefore, for more than a handful of species, or more distantly related species, this approach is not going to work at all, and therefore there will not be a straight forward approach.