Dear Dinum,

if you like to continue with traditional methods you can simply clone the PCR products to separate the different alleles. To do so, you might need an S1 lab. In the end, if you align the sequenced products, you hopefully see the differences of all alleles for that gene. Of course you need to be aware that there might also be pseudogenes or paralogs you get, in case the primer region is still present in all of these copies. But usually you see the differences in the subsequent alignment. If you would like to use more advanced techniques, target enrichment with some available bioinformatic pipelines for downstream analyses of polyploids might be an option. But this would be of course rather an overkill in case you are simply interested in one particular gene.

If you have a small number of samples and just one gene you are interested in, I'd suggest the "clone and sequence" approach. You'll have to sequence a lot of cloned products since you'll want multiple sequences from each allele, just to rule out any sequencing artifacts.

But once you have the sequences, you could possibly design RFLP markers or similar to differentiate between the allele variants.

Good luck!

Thanks Natascha Wagner & Katie A S Burnette . Yes. I am trying to do simply clone the PCR product and sequence. I went through more than 30 colonies. But still missed one allele. Hope I need to screen more colonies.

Hmm, are you certain that your tetraploid doesn't have 2 copies of one of the alleles? AABC as opposed to ABCD?

Pyrosequencing is very good for this kind of sequence but not commonly available. Sanger sequencing is not stoichiometric but if you amplify across the polymorphism to get it in a region of strong sanger signal strength( say at 100 bases from the sequencing primer) then I would expect to see a 25% signal or 50% signal from the minor variants compared to the common allele particularly if you can get a clean amplified band and a low background in your sequencing

Paul Rutland yes. its true. sanger sequencing missed the important allelic difference in the first and later part of the gene.

Yes it can happen in 2 ways with pcr and Sanger sequencing.

In the pcr stage if one chromosome has a polymorphism near the 3'end of the amplification primer then that chromosome will amplify badly and the change on that chromosome will not be amplified.

or if the polymorphism is under the sequencing primer at the 3'end then the enzyme cannot read off the mismatched primer and chromosome and the change on that chromosome will not be sequenced. You should check your genome for snps under the primer positions. If you had many results like this I would say move the pcr primer by 20 plus bases as a way of getting round the problem.

The fact that it missed 2 base changes in this case would suggest that both changes were syntenic on the same chromosome