SOS: Looking to PCR and sequence alternatively spliced PCR amplicons from a gene with 19 isoforms.

In simple terms, I have found a mutation in a gene called ATP11A. This mutation is a substitution from a G to an A at the end of an exon. According to standard splicing rules, it is very likely that this will cause alternative splicing. I decided to do reverse transcription PCR with primers that flanked my splice site mutation. There is evidence of alternative splicing because I see multiple PCR amplicons that are specific to the affected individuals that are heterozygous for a suspected splicing mutation.

This is where I am running into issues. Human ATP11A has 19 different ensemble isoforms, which is why I am picking up so many amplicons. I wish I could just sequence this, but that isn’t possible with multiple amplicons. I thought I should TOPO TA clone these amplicons, but they are so close together and it makes it very difficult for gel extractions. Now what I’m thinking is that I should try to do an RT-PCR, but use allele specific primers in my PCR master mix. I have never done a PCR that uses allele specific primers and was looking for advice about how to go about doing this? I mostly do touchdown PCRs with primers that are 20bp in length, but I am coming across protocols that are using primers that are 25-30bp long and apparently require a significant amount of optimization. Also, I see some people using forward primers with a mismatch on the 3’ end that is specific to the allele of interest, while other people introduce a mismatch 3bp from the 3’ end of their forward primers to increase specificity. Any recommendations on proven protocols would be greatly appreciated. I am aware of the primer design tool (http://primer1.soton.ac.uk/primer1.html), but I’m wondering how it works. Do I just go with default settings? Or do you change these parameters?

Do I need to do ARMS, using non-specific outer primers and inner primers that are alleles specific? Or could I just do a single PCR with a non-specific reverse primer and an allele specific forward primer? If anyone has any suggestions, it would be greatly appreciated! Does anyone have a validated protocol? Since I don’t know the exact sequence identity of my alternatively spliced amplicons, would it be safe to just assume partial intronic inclusion for primer design?