Hi again Cameron,
Also, the 19bp deletion in roy is actually in the mpv17 transcript (due to mis-splicing) not in the genomic DNA. To my knowledge the mutation itself has not been identified, so you’ll have to use non-complementation or sequencing from cDNA to ID those carriers.
On Feb 7, 2018, at 1:11 PM, Cameron Wyatt <Cameron.Wyatt from igmm.ed.ac.uk<mailto:Cameron.Wyatt from igmm.ed.ac.uk>> wrote:
Does anyone have experience genotyping nacre mutants (mitfa^w2) by PCR?
I have pigmented offspring of casper hets that I would like to genotype. Designing primers for the roy mutation (mpv17^a9) appears to be easy enough as it is a 19bp deletion. However the nacre mutation is a 1bp substituition. I've never used PCR to identify a 1bp mutation and I've read that 'sometimes it works, sometimes it does not'.
Does anyone know if PCR works for this particular mutation (presumably with a primer 3' aligned with mutant bp)?
To save people the time of writing to suggest alternatives, I will say that I know can sequence the region containing the mutation or pair mate the fish with homo caspers instead.
Thanks for any advice.
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James Lister, Ph.D.
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