I want to design qPCR primers for eGFP so I've looked up the literature for its DNA sequence but the outcome was rather confusing; too many "GFP" sequences that I now don't know which one is the correct. Any guidance?
If you exactly know the origin of the GFP-Sequence you want to look at, maybe the company or the author might help you.
Another strategy would be to find out the similary parts of the sequence, shared by all of them for the primer design.
I think, this is feasable because most adaptations were done to adoptz the codon usage of mamallian cells and of course to increase the brightness. Latter should mostly affect the chromophore domain.
If you don't succeed in finding 100 % homologous parts in all the sequences, you could try to locate possible mismatches at the 5-prime end of the oligos and play with the annealing temperature.
good luck!
There is a lot of different variants of GFP on the market now, most of them are just a single nucleotide or single domain modification comparing to the original one.
Some sequences stay the same, so you should be able to design primers for this specific sequence. If you know what company this GFP of yours is coming from, that would make your life much easier.
Here is just to help you understand why there are so many GFP variants:
go to: "The Fluorophore of Green Fluorescent Protein"
http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/pps99.cryst.bbk.ac.uk/projects/gmocz/gfp.htm
http://www.ncbi.nlm.nih.gov/pubmed/?term=Engineering+and+characterization+of+a+superfolder+green++fluorescent+protein
http://www.ncbi.nlm.nih.gov/pubmed/?term=green+fluorescent+protein+is+lighting+up+fungal+biology
Hope this helps! :)
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