If you have the sequences of both the genes from the genus, then I would nucleotide BLAST them to see the extent of similarity (protein similarity unfortunately does not always translate into DNA similarity). Based upon the aligned sequences you can pick likely candidates for primers. I have previously used this to make mouse/human/rabbit "universal primers". There are likely to be individual base differences. These can be accomodated for by designing a redundancy at the site of differences eg if in one insect the sequence has a C and the other one a T, in the design stipulate C/T at that base xxxC/Txxx for synthesis. Half your primers will have C and the other T.

If you do not know the other insect sequence, then I would try several different annealing (lower) temperatures to accomodate any internal base differences. You can get away with 2-3 base differences provided that they are not at the 3' end.

Ian

I agree Ian. I have used degenerate primers to perform RT-PCR and subcloning for the PGF2a gene from xenopus laevis in which I used the genome sequence from xenopus tropicalis . Below is a citation that describes the theory and applications of degenerate primers. J Comput Biol. 2005 May;12(4):431-56.The degenerate primer design problem: theory and applications. Linhart C, Shamir R.

Dear Rohan and Ian,

Thanks for answer. I fortunately or misfortunately new in this field so taking some time to understand especially when Rohan talked about the SNPS. The species i am studying are very close to each other and I successfully did the RT-PCR and done sequencing from four species. The similarity is 94% minimum with reference gene from NCBI and both the starting and end areas are well conserved. Actually one species i have selected the same as NCBI to have a model for my work. The model gene is working well and i am getting expression and WB is ok. But other three genes no response at all therefore, I am bit worried. Please suggest what should i do.

FAHAD

Dear Johnny Sena,

I got the RT-PCR successfully and the sequences are well conserved. So i still need to design degenerated primers or need some optimization to get expression

FAHAD

YOu can do the nucleotide blast using the primer sequence . If you find SNP in middle of the primer sequence then it is ok. If you found any SNP at the 3 end or near the 3 end you need to design new primer.

Dear Fahad,

If the level of similarity you refer to is for nucleotide sequences, most primers you design will work for most species. However, third codon position are the most variable. Designing primers that end in a first (or second) codon position increase a lot their chances to work in multiple species. If you have trouble with a certain gene, I would try designing a new primer pair, and combining the different primers.

Once you have amplify the product I would sequence each species and obtain either specific primers for each or "universal" primers in a region identical in all species. However, I am not sure about what you mean by "to get gene expression", so not sure if this optimization will always be necessary.

Hope it helps,

Bàrbara

From your question I understand you want to RT-PCR the product of this gene to make an expression construct to study the protein? If you want to make sure that the start and stop of the cDNA do not have SNP's that might not influence the RT-PCR but can affect the protein: I would try to PCR the start and stop of the gene on gDNA level to see what the actual sequence is. Then make primers at the start and stop and RT-PCR the gene of interest. If you make primers that are similar but not exactly for that species you are altering the sequence without knowing because your RT product contains the primer sequence but not the "real" sequence. Another option could be that other ATG's upsteam might be present that add or alter a piece of peptide chain, possibly important for only this species.

Thanks to all and i will try to combine all suggestions and try to work out.