Hello,
I'm trying to introduce mutations into a gene via CRISPR/Cas, and I have to check the presence of mutations in a short DNA segment (about 20 bp) within a 512 bp long sequence. Unfortunately, this DNA is a GC-island. After many attempts, I managed to amplify it with DMSO in the reaction mix and a semi-quantitative PCR protocol lasting 3.5 hours. The amplification product was controlled on agarose gel, and it is good.
I have many clones to analyze and would like to save the cost of a sequencing service, so I'm trying to pre-select mutant clones via melting curve analysis.
Teoretically, it is possible to use a 5'-fluorescein labeled probe (SimpleProbe, Roche) to this purpose. Therefore, I mixed this probe with my amplicon, and used the LightCycler 2.0 instrument to perform melting protocols without prior PCR.
However, I did never obtain a melting curve! I don't think the error is in the temperature setting, because I started heating from 5 degrees below the melting of the probe up to 95°.
Any suggestion?
Hi Enke,
This answer is not about the high resolution melting analysis but I think it can be of use. Check out GeneAbacus kit from Countagen. It uses a combination of padlock probes and rolling circle amplification to detect CRISPR-mediated mutations. This method works very well across GC rich regions. I am up for a chat if you are interested in knowing more about the technology.
https://countagen.com/product/
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