I tried to design primers using several tools but it seems that in reality, I got only several genes after verification by sequencing of the PCR product. I feel like there is some sort of trial and error in designing a primer. I appreciate for all tips.
Do you want to design a primer that only detects one gene and not the duplicates? Do you know the duplicated genes? When I design a primer, I check the specificity using primer-BLAST at NCBI. If some duplicate, or close relatives appear then I pull out these sequences and align them using Clustal at EMBL-EBI website and look for a region that is unique to my gene and re-design primers there.
Thank you Amanda for the answer. Actually I would like to get all of the genes. I work on group of plant transcription factors which has common domain and relatively similar sequences. I tried to design in 3'-UTR to avoid conserved region and might be able to differentiate one gene from another. I used Geneious as tool, and also Primer3 in NCBI. But finally, when I did the PCR and verify the sequence, I didn't got the sequence I wanted. It seems that maybe I should use degenerate or nested PCR.
Oh, I see. It might help to bear in mind that the last 5-6 bases at the 3'end of your primer have to be specific to the TF you want, to prevent mis-priming to a close relative. Degenerate primers will be of little value here unless you don't know the sequence of your family members. Nested PCR will help though especially if some of your family members are expressed at a very low level.
hi putranto,
Did you try HYDEN for degenrate primer synthesis
what plant do you study ? and what kind of analysis do you want to perform with the primers ? (cloning/sequencing or qPCR, ...)
Thanks Amanda and Bhupendra for the advice. I did not try yet HYDEN.
David, I worked in rubber tree and I would like to do qPCR analysis. Eventually, I expect more specific primers that could represent my genes (there are hundreds of them identified in silico) but I realized it was not so easy to design the primer (I got only half of them) because some of these genes are very close in term of sequence.
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