I'm trying to develop a genotyping assay to identify heterozygous vs. wild type mice, the mutants have a 1kb deletion in my gene of interest. I've tested several primers and have a couple sets that work in a traditional PCR/gel electrophoresis, but unfortunately the mutant band is not always super clear. I'd like to have a secondary genotyping assay before I invest in sequencing. Has anyone tried HRMA on large amplicons, and can you offer any tips? Thank you!
I've used it only for small products, but you could always try it for larger. I had good luck adding in SybrGreen dye to my PCR master mix. Since your products will be VERY different sizes, you can do a standard melt curve and not high-resolution. Test out some products to see what temperature range is informative.
Also, you could set up two PCR reactions. One with primers that will only amplify a product from the wild-type allele and one the will make a product only from the deletion allele. If you know the EXACT location of the deletion, you can make a "deletion spanning" primer and treat the deletion as if it's an exon/exon splice junction. Just a thought for another strategy.
why do you need a large amplicon?
If you amplify across the deletion the amplimer only needs to be short ( 200-300 bases) to prove the deletion exists and any 200-300 base amplimer within the deletion will indicate that the wild type sequence exists
Thank you all for your recommendations! Via sequencing I've been able to find the exact location of the deletion and have been able to use primers just outside the deletion to do what both Katie and Paul suggested. The PCR genotyping is very straight forward now, I'm using two sets of primers: one set that sits just outside of the deletion with PCR conditions that only allow for a small amplicon (will only show mutant band) and a set that has one primer inside the deletion (will only show wild type band). I tried HRM as well and it worked ok, but not consistent across samples. I'm finding that the DNA needs to be stringently purified to get the best results, still troubleshooting though. Thanks again!!
- Badges
- Science method