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?
Dear Justin Pater,
TOPO cloning will be the best option. After cloning and plating, perform a colony PCR and restriction digestion. Then run the restriction digested products on an agarose gel. After picking single colonies from plates with inserts in it, the product might be pure. So, there won't be any issue of products with similar/close molecular weights. If the colony is pure, you will get only two bands (vector and insert). You can do gel purification of the insert and sequence it.
Regards,
Siva
Try to run in PAGE gel if it is not more than a kb size, or run in 3 % agarose gel having 2% normal and 1% low melting and run a long gel. Isolate the fragments and do rePCR. i think this should work
I don't think you can necessarily assume that the type of variation you depict won't affect splicing specifically at that donor site (for example by impacting local secondary structure - which might allow a cryptic upstream or downstream donor to be used instead). The most likely possibilities are allele specific splicing in the hets, with the variant resulting in: skipping, shortening, or lengthening of the variant exon.
It's a little difficult to know what's going on with your gel without knowing the specifics, but you have a clear genotype dependent difference, which makes your life easier. You don't need to fret about separating the bands - just Topo clone from the PCR product and screen inserts by size... But since you presumably know which exon is altered, the obvious first step would be to design qPCR primers spanning that boundary (the 19 isoforms is irrelevant, only the number of alternative 3' exons to which that donor site splices).
You're presumably working from blood samples, so ENST00000375630.2 is likely the one you pick up
(GTex is your friend: https://www.gtexportal.org/home/gene/ATP11A).
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