I’m currently trying to develop a 10 SSR marker multiplex for allelic screening using the universal tags and the method (iii) from Blacket et al. (2012 - DOI: 10.1111/j.1755-0998.2011.03104.x ). In which every locus/target SSR region has a reverse primer, a forward primer with a tail that corresponds to a universal tag with a fluorophore attached. Therefore, each locus has 3 components to amplify it, and the 4 universal tags used are shared between 2-3 loci each.
So far by following the protocol in the paper it has resulted in very diverse peaks sizes in my electropherograms, generally extremes. With some peaks too large to be read properly in GeneMapper v5 (Exceeds the max detection) and others barely distinguishable from the background noise. I think this is due to some primers being more competitive for the universal tag they share, as all have amplified each locus fine individually.
My ‘solution’ was to only change the Forward (F) primer concentration but keeping them all below the overall universal tag concentration to hopefully exhaust them during PCR as this 3-primer system requires. Specifically, for each shared universal tag, increasing the F primer for the less competitive loci primers and decreasing the F primers for the more competitive loci primers.
However, only changing the F primers has generally reduced the size of the peaks produced from the more competitively amplified loci, and all but removed the peaks produced from loci targeted by both the less and moderately competitive loci primers. I assume that this 'solution' did not work because I did not adjust the other corresponding primers for each locus, but I am not entirely sure how to adjust them so that I get clearer peaks to analyse.
I have seen other papers that reference Blacket et al. (2012) mention different primer concentrations than what the original paper recommended but I have not seen anyone mention the problem I have above, nor in detail on how to adjust the primers, even in the original paper. Which is why I have asked this question.
Is another option now that you have a feel for how intense the peaks are either run 2 dilutions of the amplimers,,,,low dilution to get the weak peaks with strong peaks unreadable and a high dilution to get good definition of the strong peaks but nothing on the weaker signal, Alternitively if this is a multiplex pcr run 2 multiplex pcrs one with strong amplifiers at N pcr cycles and another for weak amplifiers at N+4 cycles then mix the 2 products
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