Dear Syed,

That is not an easy problem to solve. Your suggestion of doing a multiple alignment and identifying the consensus region is certainly a good one, but this way you will fish out all sequences using PCR and you still will not know which is the one you are interested in. You can also start from the protein site. If it is an enzyme you can think of purifying the protein and characterize it (substrate specificity, Vmax and Km). Once you have the protein pure, or have identified its protein band on a gel, you can take that and do a sequencing by mass spectroscopy. With that information you can go back to the nucleotide sequence and identify unique elements of your sequence in the above multiple alignment.

I think, You can check translated sequence of all genes and find the specific domain as per your requirement. You can do this in NCBI Only. Hope this work

Align and puck a common region with identical sequence to design primers

Syed Monzur Morshed As a student, there is not much to experience around. Yes, I would just design primers from the conserved regions for PCR of your own species. The 21 similar sequences you mentioned might be a total from all different species? Your own species might have much less than that? Try and find out.

Dear Professor Fred R. Opperdoes , Thanks a lot for your suggestions.

Dear Dr Kailash Lipne , Thanks a lot for your suggestions. I am doing this at this moment.

Dear Laurence Stuart Dawkins-Hall , thanks a lot for your suggestion. Right now, I am doing this. I will let you know what is happening.

Dear Dr Yuan-Yeu Yau , Thanks a lot for your suggestions. All the 21 sequence is from the same species and for the same target gene. The sequences have some differences between them.

I agree with the suggestions made

Dear Syed,

Many of the suggestions already mentioned will help. Nevertheless, I would like to add a comment to your question.

I did not understand the meaning of "correct sequence." Isn't it a "useful sequence for you in this stage"? It is essential to clarify the purpose of the experiment. Once that is clear, you can choose a more appropriate sequence among them. It is a good idea to check each sequence's source and confirm it by asking the NCBI staff what kind of index was used for registration.

Dear Dr Shintaro Iwashita,

Thank you for your comment. I have already chosen the target gene for my experiment. But the problem is there are 21 predicted sequences in my species database for that target with the same name but different sequence [With different location (LOC)].

So, I wanted to know how can I know which sequence is the correct one from that 21 sequences based on which I can design my qPCR primer. I used 'correct sequence' in that context. I hope I have cleared the point.

Please let me know If there is any lacking in my understanding of the problem. I will appreciate your kind and important suggestions.

Regards,

Syed Monzur Morshed

Dear Syed,

Thank you for your question@. I just wanted to tell you that the issue is the species and tissues and developmental stages (embryo or adult). As you might know well, one critical issue is stage-specific splicing. By imagining the obtained quantitative results in the sample's time and space, the candidate sequences can be further narrowed down. I hope this perspective will be useful for your research.

With my best regards,

Shintaro