3-primer PCR Genotyping - problem in hemizygotes - (Feb/06/2015 )

Hi everyone,

I'm new here but desperate! I am using a 3-primer PCR to genotype knockout animals, with forward primers for the wild type and mutant alleles and a common reverse primer. This has worked well for a few litters but I have suddenly encountered a problem. The mutant band in my hemizygous animals has completely disappeared (I always run a known sample for both homo strains and a hemi.) I ran separate 2 primer reactions for each combination and found that the mutant primer pair amplifies efficiently in the homozygous knockouts, but poorly in the hemizygotes - I do see a faint band but not enough to confidently interpret in unknown samples. This band cannot be seen in the 3-primer reaction. The wild type primers work equally well in homozygous wild types and in hemis.

If anyone has encountered this before, I would love to know why it's happening. I'm at a complete loss!

I extract my DNA from ear notches using the Qiagen kit.

All 3 primers have T m of 66-67 degrees and my annealing temp is 60 degrees.

I use Qiagen HotStar Taq and its standard buffer.

I have ruled out reagent and freeze-thaw issues by using completely fresh reagents.

Thanks in advance :)

Are the fragments significantly different in size? Short DNA fragments amplify much better than longer ones. You can always do the reactions separately, but if the combined reaction is necessary, you can try optimizing the annealing temperature with a known heterozygous sample.

Thanks for the response. Yes, the WT fragment is 200 bp vs 600 for the KO. But the KO fragment amplifies very well in the homozygous KO animals, that's what I can't explain! Anyway, I dropped the annealing temp to 58 degrees and increased the MgCl2 concentration in the reaction. I got way more non-specific bands but I can now identify the genotypes successfully - hooray!