Your question is very interesting. I agree the prime hypothesis is the lack of specificity from the primer but this doesn’t explain 1) why this is specific to NgAgo only and I’ve never seen this with CRISPR or normal genotyping reaction using the same primers? 2) This occur with all the primers I used for NgAgo for 3 different genes, (not only the one I show on the post) 3) The primers pairs match in fact perfectly (after reanalysis of the chromatograms, the mismatches were due to scoring error from the sequence analysis program. More than happy to release the sequences) with the sequences amplified but not with the mouse genome sequences. It requires a minimum of 4 base pairs overhang from both ends with the genome to amplify. I am still puzzling how does it work but I don’t think the pairs have integrated into the genome. Could be a ligase or eventually a nickase but I how a nickase would work without any specific recognition sequence (doesn’t match at all with the 5'P oligo). BTW if you have a better idea than mine, more than happy to consider it.
The DNA extraction method was lysis buffer + prot K and heating at 95ºC. It should have eliminated all possible proteins but NgAgo might be resistant to the heat. We are checking if a proper inactivation of all proteins by phenol would prevent the presence of these bands on a gel.
Regarding the 5'P oligo, the sequences I used as a 5'P don’t match at all any of the amplified regions. Importantly I have injected 2.5 ng/µl of oligo and cultured the embryos to blastocyst for 4 days and I have more extra bands than 25 ng/µl 5'P. I don’t think the 5'P oligo play a role here. We are treating the DNA too to verify this hypothesis.
In short the story is far from over and we still have few experiments to do to understand how does it works.
More than happy to get your feedback.