I want to detect insertion/deletion polymorphism of 5 bp with PCR with two primers. Detection is on polyacrylamide gel. I always detect just homozygous insertion or heterozygot with one unspecific but totally replicated band of 180 bp (my bands are 126 and 131). I cannot get homozygot for deletion. Has anyone had the similar problem? The MAF is good enough to expect at least 20% of homozygots for deletion genotype, so it shouldn't be a problem.
may be you can find a cut sequence for a endonuclease in your Insertion and diggest the PCR product. Than you have 3 bands at heterozygot, two bands with insrtion and one band without....
Focus on the reason for misamplification. Maybe there is a new primer binding site causing the amplification of 180bp (e.g. a second insertion around or unknown polymorphism generating a new primer binding site, ... could be linked to the deletion, maybe compensatory,...).
It might be helpful to sequence the 180bp amplicon.Therefore, it could be useful to clone the amplicon into a vector, transform it into bacteria and prep it clonally. If successful you will know what kind of sequence you have (by blast) and that will help you to identify the reason for the problem. I hope...
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