Allele specific PCR (ARMS-PCR) works so much better and is more reliable when 2 different SNPs are targeted at the same time. Otherwise, results might not be reliable.

On the other hand, what you actually see is different performance of each primer pair, which is a basic feature of PCR, even if you keep 1 of the primers constant and the allele specific primers differ only in 1 base

Thanks @Iker.

Actually among two forward primers, one is for say C allele and another is for T allele. If i target one SNP (C/T), I do PCR with Forward primer C or T allele specific and common reverse primer. Already told that both primers differ in 3' end C/T, on that case what is the resolution of my findings??. Say i got light band for C allele and bright bands for T allele on the same sample.. Can i say itz for heterozygous?? If yes, why they are not equally Bright even when PCR conditions and compositions are remained same?? 

Again at 3' end of the primer where DNA Pol binds, Do you think that DNA pol misses the right point to amplify which results light or no bands on gel?? That's why I came with the reliability.

Thanks @Roberts for your sum of the great suggestions.

Actually I was much more careful about the designing of the primers. And only one SNP was targeted as per PCR reaction. 

Thanks again.

I would recommend adding a -2 change in the primer as in Tetra-primer ARMS PCR (Ye et al. NAR 2001) since this will give further specificity to your primers. This is because although you have a mismatch at the end of your forward primers (for the two alleles), sometimes PCR extension occurs and you get faint bands for the absent allele and you would erroneously call that sample as heterozygote.

And Yes! The combination of alleles you have at a SNP does matter! Transversions are easier to detect than transitions (see Ye et al. for more details).

Even if you want to use your current primers, then always add positive control samples for your two alleles in 'every' run. This will help you to decide whether alleles are present . If you don't have positive controls (e.g. from sequencing) and your SNP is common (MAF > 0.05) then you could make a pool of let's say 20 samples and genotype that sample in every run.

Final QC step: Do HWE exact test (or Chisquare if genotype counts are above 5) and see if there is significant deviation. Allele specific PCR usually results in an 'excess of heterozygotes'.

Hope it helps.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55900/

Hi

I too suggest you to incorporate -2 mismatch at 3' end of your forward primers. This will eliminate your unwanted amplification. Check the gel pic attached where I have designed ARMS primers using similar approach and got desired result. 

Good luck!

Thanks for the idea @Maroju,

I do appreciate your practical experience of adding the -2 mismatch but tagging some oligos in 5' of forward primer can put me in more trouble. Specially in case of Annealing and again i cant also delete the consideration of non-specific bindings.

My target was not the non specific bands, it was the intensity of bands because there was similar size bands i cant ignore by calling it non specific.

Thanks again.

Dear Rahman

As you can see the 5' tagged bases are completely unrelated to template sequence therefore doesn't hamper your PCR by unwanted annealing. I used the term non-specific for your low intense product of similar size not in the general context.

Thanks and good luck!!