I've been trying to genotype het mice with little success. I initially started with a protocol (provided and tested by the mouse strain creator) using nested primers (FWD, mutant REV, wildtype REV) targeted at the specific gene and could not visualize the mutant PCR product in het offspring -- WT and mutant homozygotes gave successful, respective DNA bands. To avoid allele-specific competition, I performed separate PCR reactions for the mutant and WT alleles and got the same result -- het offspring only showed positive for the WT product (and both reactions were successful). I even went further and designed new primers+reaction for the neomycin cassette used to generate the mutant allele -- the mutant homozygotes show a PCR product for this, but again, the hets do not. What else can be done to genotype for heterozygosity? Is it possible that the mutant allele, when in competition with a WT version, can be so silenced/repressed that it affects PCR? (I very highly doubt that all 8 of the absolutely-for-sure het offspring had recombination events in this gene.)
it is not very scientific but while you are waiting for a good answer to this problem borrow a different polymerase ( preferably hot start ) from another researcher and try the het pcr. I have occasionally found some Taqs to be quite fussy while others will amplify a sequence without any problems
I don't think it would be the case, since gene silencing/supression is not the genomics phenomenon, but transcriptomics. The gene is still intact and should not effect the allele specificity.
What is the size of your WT and mutant PCR products? When using all 3 primers, do you double the amount of the common FWD primer? If het offspring only give teh WT band, how do you know they are Hets?
Gregory Dressler, thank you for asking -- The mutant product is ~350 bp and the WT product is ~150 bp. The reaction using these primers as a set DOES include doubling of the FWD primer. Our breeding strategy involves crossing homozygous mutants to homozygous WT (verified using this PCR method previously; parents comprise the controls for the offspring). We're specifically using this breeding strategy to eventually achieve a full het breeding scheme to obtain all three genotypes in each litter, so we have to be able to verify hets.
I cannot think of a definitive reason why you cannot detect the mutant band in hets. The fact that you cannot see this 350bp band using just the FWD and MUT primers is odd and suggest that this reaction is not optimized. In the Homozygous mutants, how strong is your band? How many cycles? Perhaps having 2 copies makes the band appear with fewer cycles. You might try adding 2-4 additional cycles of PCR. Also, lengthen the elongation step (2 min.?) as larger products are always amplified with lower efficiency.
Gregory Dressler -- The homozygous mutant has a beautiful, bright band at 350 bp (whether the reaction is done with all primers or just the FWD + mutant REV). I've played around with the PCR settings (annealing temp, elongation time, cycle number) in my alternative, neomycin cassette strategy but not with this gene-specific primer set. In regards to genotyping for the neomycin cassette, optimizing the reaction did not resolve the het samples even though the homozygous mutant product lit up even more. Do you think that the gene-specific PCR reaction would give successful results with optimization even though the other strategy did not?
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