Designing primers for your high resolution melting is easy enough. A quick run with some known genotypes …. and look for the melting curve differences among samples. Too simple. But what about the temperature-neutral SNP’s? The what?
A number of SNP’s ( ~ 4% in the human genome) have no affect on the melting temperature of an amplicon – that is, wild-type can not be distinguished from homozygous variant. In short, small amplicon HRM will not work. Well, not immediately.
There are a couple of modifications that can be done but one that we like using in the lab is snapback HRM. Yes of course it’s by Carl Wittwer’s team – how did you guess? We could describe it but check out the paper here and see figure 1. Essentially a 5′ probe elemnt on one of the primers folds over (snaps back) over the SNP during the melting phase to give better discrimination than the amplicon melt.
The beauty of the method is that you get to use both the amplicon melting (scanning for unknown mutations) as well as better SNP discrimination. Below are some images for a method we developed on the Mic qPCR cycler to look for 3 tightly clustered mutations in varroa that confer resistance to common treatment chemicals. The most common mutation (L925V) cannot be distinguished from wild-type sequences – but the snapback element in the 2nd picture (melting at lower temperatures ) easily discriminates the two.
So for those mutations that prove tricky, especially class IV SNPs, consider snapback HRM. For a bit of mental soduku, you can design probe elements yourself. Or check out: https://www.dna.utah.edu/usb/snap.php