I am performing a KO of my gene by frameshift in zebrafish. I have been screening this by the disruption of a restriction enzyme site in a PCR fragment. I now have an incross of 2 mutant fish and am hoping raise the progeny and get some homozygous fish. I will use DNA from tissue sample as the PCR template but I am wondering how I can separate homozygous from heterozygous. In my experience so far the F1 generation from an outcross with wild type showed an extremely faint undigested band (wt DNA) compared to the really bright digested band (indicating mut DNA) for some reason even though an outcross should automatically result in heterozygous fish containing both wt & mut (digested & undigested) DNA. I presumed that the concentration of the mut & wt allele would be similar. Has anyone come across this difficulty before. Could there be a reason a mutant would amplify more in a PCR reaction (it is only 7 bp shorter)? Any suggestions for a way to tell the difference? I am concerned since the band is so faint on heterozygotes I may misidentify heterozygotes as homozygotes.
Hello Amanda,
maybe you could try the Surveyor assay, where the surveyor nuclease detects missmatches in the DNA and cleaves it at that specific position (heterozygous alleles should be cleaved therefore, whereas homozygous should not).
http://eu.idtdna.com/pages/products/reagents/idt-surveyor-mutation-detection-kits
Thanks for the suggestion. This the same as the T7 assay yes? I could screen the fish with both and compare the results I suppose. At least until I know what the results should look like in each.
Ok, I wasn't quite sure what you've tried before. T7 and surveyor are similar. I just can tell you, that it worked well for me to distinguish between homozygous and heterozygous. You could additionally sequence your amplicon and have a look at the chromatogram.
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