I am doing multiplex PCR for microsatellite population genetics study. I have 4 flourescent dyes (FAM, PET, VIC, and NED) with 3 locus pairs each PCR run. Out of my 400+ samples, there are 50+ samples which doesn't produce any bands in the gel from the NED group. I already did gradient annealing temperature, changing the reagent concentration but still there are no bands in this samples. However, these 56 samples have good bands for the FAM, PET, and VIC so I don't think the problem is my DNA. Thanks.
In some cases, some of the amplifications are more efficient while other work less efficiently. In such cases, the relative concentrations of primers for each locus must be altered to compensate this effect. A second and less probable option is the existence of null alleles, that is alleles that can not be amplified by a particular pair of primers, mainly due to lack of annealing. This makes that homozygous individuals produce no amplification ( we have found this problem in some sheep populations, for instace).
My advice in your case is making a simplex PCR for the problematic samples in the questioned loci. In case PCR works this way (check it in agarose gels), you may add the PCR product to the multiplex product prior to capillary electrophoresis to get the results you need in a fast way.
Hi Maria,
If I got you correctly is that you have a set of microsatellite primer pairs, and for each pair the forward is end-labelled using one of the four dyes (i.e. FAM, PET, VIC, NED). And from your amplifications, only the primer pairs that are end-labelled with NED do not amplify.
Based on your PCR results, I do understand that the problem may not absolutely depend on DNA because same samples do amplify on other loci. I passed through this in the past and two possible reasons could be invoked: i) the problematic primers may require disproportionately high DNA concentration, and ii) As already mentioned by Luis (see above) there might be some mutations that occurred at the primer binding sites of your problematic primers. And, this is more likely if the primers were designed from populations that exhibit massive genetic differences with your samples.
To troubleshoot, you may have to PCR amplify your problematic primers separately using more DNA (for the case of low DNA concentration). Alternatively, you may have to lower the annealing temperatures for your problematic primers (but remember this will permit unspecific priming). If they do amplify, you may have to do a post PCR multiplex approach prior to genotyping. Otherwise you may have to discard those primers
You got a funny case here. If there were some null allele, it is unlikey that all these 56 samples had mutant at all the three loci. Similarily, if amplification efficiency of some primer is comprimised by the primers of other loci, it will not explain why you had no band for either of the three loci.
So you may like to run the NED group and also some reactions with the individual primer-pair simutaneously as control. If you had band for those with individual primer-pair, then you will probably just rerun the PCR using individual primer-pair for these 56 samples. If you still got nothing at all...then you got a big problem :)
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