I am working on gene sequencing for an organism that has little to none sequencing done. I have acquired the homologous sequence for that particular gene across three species. I would like to know how do I pick the coding region of this gene to design a primer for the RNA sample that I have obtained from my sample.
Hi Preetha, I would suggest comparing the gene sequences from the three species and designing the primer at the region of maximum homology. Even if there are a couple mismatched nucleotides, the primer should still bind. If you are unsure of where the coding region is, you can view the complete sequence at NCBI gene or NCBI nucleotide, find "CDS" on the sequence page and it will highlight the coding region. If the CDS is similar in the three other species, it probably will be in your organism too!
Design a few sets of primers just in case the first set doesn't work. Include the genomic DNA from your organism as a positive control as well. It will make a larger product that includes the exons & introns. If nothing else, you can clone and sequence the gene & then you'll have a chance to redesign primers for the RNA.
Good luck!
When you don't know anything about the sequence and you are trying to design the primers based on homologous sequences from the related organism you should go for degenerate primers.
First thing you should check is: the conserved domains (use NCBI CDD for this purpose, it marks all the conserved domains in the submitted sequence) in the protein sequence among the homologs and you can design the primers from the corresponding nucleotide regions in these conserved domains. At some places nucleotide bases may vary in conserved domains, you should include degeneracy at these places in your primer, it works.
If data for your protein conserved is not available in the database, then you can follow the suggestions of Sarrah Lahorewala.
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