I am using ARMS PCR primers, have tried it 1. All four Primers together, 2, outer primers + inner for one Allele and 3. only one outer primer with corresponding outer primer.
1. Control primers perfectly amplify,
2. One allele perfectly amplify when present
3. but the minor Allele in any case (2 primers, 3 primers and 4 primers) doesn't amplify at all. while the minor Allele frequency is 0.2-0.4, where is the problem? the primers QC was/is fine, in-silico also fine, annealing tried at various temperatures and tried the best (brightest band's) annealing but no avail, help please
try running a gradient of annealing temperatures for the non working primer on a sample known to have the minor allele. Your annealing temperature may be too high or possibly in the case of your samples the minor allele is also linked to another polymorphism situated under the primer making the annealing temperature lower than expected
Hi Muhammad Zakria
May I know if you have tried the gradient annealing temperature with all of the primers in one reaction? If not, I suggest you to do so to find the best annealing temperature not only for the each pair of the primers but for all 4 of them in one reaction. After you got the optimal annealing temperature you can do optimisation on the ratio outer to inner primers concentration.
I hope this suggestion help in your research. Good luck!
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