We used CRISPR-Cas9 to create mutant zebrafish by injecting it into the eggs of wild-type (WT) fish at the one-cell stage. After letting them grow for three months, we cut their fins, extracted DNA using the NaOH method, and ran the samples on a 20% polyacrylamide gel to estimate the level of mosaicism.
Now, we are crossing these F0 fish with WT fish to check which ones carry the mutation in their reproductive cells and can pass it to the next generation. We extract DNA from larvas and after PCR for our gene of interest, we run it on 3.5% agarose.
We expected to see either one band (only WT) or two bands (WT and one mutation). But in some offspring, we’re seeing three bands, which has left us confused.
Could this be a technical issue with our method, or does it mean that some fish have more than one type of mutation in their gametes? (pictures are inverted display of 3.5% agarose, our gene of interest in 204kbp)
I think that you are seeing the consequence of having a snp in your pcr amplimer. Thus there is no size difference between homozygotes NN and MM which is the strong large amplimer. With good pcr amplification however the NN and MM amplimers melt and then reanneal before the primer can anneal in the last round of pcr. Now we have a heteroduplex of NM and MN which has a bubble in the double stranded sequence and this makes the amplimer run slower ( looks larger) through the gel giving your weaker largest band.
You can test this by isolating the lower band of a sample with 2 large bands and heat and reanneal this band.It should ,once again, form the 2 large bands that you see in this gel. Or by sequencing the lower large band where there are 2 bands
Thank you so much for your explanation! It helped clarify the concept, especially regarding the heteroduplex formation.
However, it raised another question in my mind: if the heavier-looking band is due to heteroduplex DNA, how come we also see it in samples like 13 and 15, where we don't observe a separate mutant band? Wouldn't the heteroduplex require the presence of both WT and mutant strands to form?
Also, for the test you suggested—do you mean we should cut out the upper (heavier looking) band from the gel, heat it to denature the strands, and then reanneal it? Would we expect it to resolve into two distinct bands if it's indeed a heteroduplex?
Thanks again for your help!
Sorry Sadaf I have confused you with my terms mutant and normal. These do not refer to your smaller band but refer to the alleles of a snp found between the pcr primers in the larger (middle and strong) band . This snp does not exist in the smallest band but only in the large band. So if the alleles were A and C then the strong middle band is a mixture of AA and CC which both run at the same size and the heteroduplex is A+C which forms a bubble in the DS dna causing it to run slowly creating the largest band
Yes cutting the strong band out and denaturing and reanneaking should once again form 2 bands. Or sequencing would show 2 sequences if the strong band was sequenced
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