I’m tring to reproduce a ASO-PCR able to discriminate between two different strains. The ASO is due by using three different primer: a forward one and two reverse pair; the former reverse is able, with the forward, to amplify an internal control, while the latter reverse gives an amplicon shorter than the previously one but able to discriminate between the two different strains. So the discrimination power of the PCR is due only by the second reverse primer. The problem is these strain differ from each other from only two pair of bases. I tried a lot of experiments but so far I was not able to discriminate, I mean, the two PCR are positive with the same sample, so I can’t cluster my samples. Does somebody know a good site having informations and troubleshooting about ASO-PCR?
Many thanks in advances.