I am trying to identify these 3 genes among some tomato cultivar collections and after aligning some sequences from NCBI, I couldn't find unique sequences to target for specific primers. There were just some single nucleotide differences at some positions. I just don't know how to go about it.
Hi there,
What you could do is to look if these nucleotide substitutions have an effect on the restriction map of the gene. If so you may use RFLP technique.
Just an example for illustrating the use of PCR-RFLP:
https://www.researchgate.net/profile/Yu-Huei-Cheng/publication/51423356/figure/fig1/AS:601703342669840@1520468664478/Principle-of-PCR-RFLP-A-Natural-RFLP-The-rat-rs8144801-with-genotype-A-G-is-used-as_W640.jpg
If you are trying to design primers that will work to amplify these genes in multiple cultivars, a single SNP will not ruin a primer's ability to anneal. Try to put the SNP towards the 5' end of the primer and you might have to optimize the annealing temperature to get efficient amplification.
If you are trying to design primers to differentiate very similar genes, you might have to use dCAPS or RFLPs or just Sanger sequencing.
Good luck!
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