I have coordinates for my genes of interest and I want to design primer set to identify single mutation. I got the DNA sequences using UCSC browser and designed my primers using Primer3, however, all the primer sets that were provided does not include the position I am interested in. Can I get some suggestions on how to go about it?
Thank you
It depends if you are amplifying the sequence in order to sanger sequence it in which case design your amplification and sequencing primers about 150 bases either side of the mutation because you get no sequence under each primer or for the 30 bases just inside each primer.
If you just want to show that the mutated sequence exists then there are 2 good possibilities. If the change created or destroys a restriction enzyme site then amplify as before and cut the amplimer with the appropriate enzyme,
If the change does not create a restriction site then consider using the DCAPS technique https://www.ncbi.nlm.nih.gov/probe/docs/techdcaps/
where adding a second change to the amplification primer almost always creates a restriction site then you can amplify a short template and cut it to prove the existence of the base change that you are interested in
dcaps design is at http://helix.wustl.edu/dcaps/dcaps.html
Well, I recently started to work with the AlleleID software, there they have some tools to identify mutations and everything that you need, they design just for what you want. They even have a tutorial to teach how to work in the program and have 15 days to use for free if you have an institutional email available.
http://www.premierbiosoft.com/index.html
here you can find it. Hope it could help.
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