Nacre primers

[Cameron Wyatt]

Dear all,

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.

Regards,

              Cameron

--

Cameron Wyatt
Zebrafish technologist and manager
HGU fish facility
Institute of Genetics and Molecular Medicine
University of Edinburgh

[James Lister]

Hi Cameron,

Actually w2 is a substitution that fortuitously creates a Dra I restriction site, so the genotyping is pretty straightforward and you can use pretty much any primers to your liking that flank the mutation (although be aware there is another Dra I site not too far away.)  I can send you our primer info/protocol if you like.

Cheers,

Jim

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————

James Lister, Ph.D.

Department of Human and Molecular Genetics

Virginia Commonwealth University School of Medicine

Richmond, VA 23298 USA

James....@vcuhealth.org

[James Lister]

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.

-Jim