We are studying a gene with 5 exons. In a patient with a mutation in one of the introns we detect a splicing defect such that exon 3 is missing (exons 1,2,4,5). Yet we also detect the wild type variant containing exon 3 (exons 1,2,3,4,5). I can design an oligo that spans the exon 2,4 junction to measure specifically the variant that lacks exon 3. But if I want to compare the relative levels of the two variants in the same cell by qPCR, how would I do that? Oligos that anneal to all other exons will be in common between the two variants.
if the primers could span exon 2 and 4, you shoud be able to get two bands. upper band with exon 3, lower band without. calculate the ration of two bands could give you a hint about the two variants.
Hi Mingming, Sorry, but I don't understand your answer. If the oligo spans the junction between exons2 and 4 how could that give me a second band representing the variant with exon 3?
if I understand the question rightly, I think in your template you should have two variants. one with exon 3 and anther without. If the primers could be degined to bind to exon 2 and 4, both of the variants should be obseved. By the way, I do know the size of the exon3, if it's not too small, it should be easy to sperate the bands with agrose gel.
I see. We did that already. I was not clear enough in my question. I wanted to know if there was a way to do this by qPCR. We have run the gel but that is not quantitative enough. Sorry for the confusion.
Maybe you could degin a pair of primers targeted to exon 3, and another pairt targeted to other exons.
I'm thinking the melting curve might be the way to go. The two species should be different enough such that they have different melting curves. But again that only measures the endpoint, not during log phase amplification, so it may not be quantitative.
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