Do multiple double peaks surely indicate both alleles of my PCR product are sequenced? (Sanger)

Question

[michael.nagle]

I sequenced PCR products with primers expected to amplify both alleles of a hybrid. I see double peaks and believe these indicate positions where there is a SNP. I need to be sure that both alleles are sequenced because I need to select target regions that are completely conserved between the two alleles.

Is there anything other than the presence of two alleles in a PCR product that could cause the appearance of double peaks?

Thanks

Answer 1

[John]

There are quite a few things that can happen that can lead to "double peaks", but you really need to look at the sequencing trace to be sure/rule them out. Off the top of my head so no comprehensive list: non-specific primers, chromosomal rearrangements/CNV, psuedogenes, contamination with another similar species (if it's gut biota or similar), poor quality Taq Polymerase that introduces errors. However Sanger sequencing is still considered the 'gold standard' for this sort of thing, and you can work around a lot of these issues with modifications to the wetlab protocol.