I need to design primer for two paralog genes, one of the gene doesn't have any unique region, Can anyone suggest how design the primer for real time PCR for this gene.
Thanks
sometimes even a single base polymorphism/snp is enough.If the mismatch base between the 2 genes is at the 3' end of the pcr primer then there will be enough of a difference in annealing temperature to allow one allele to amplify while the mismatched primer pair may not amplify. Essentially ARMS pcr and if you can find more than one mismatches in a primer then even better. Then it is a matter of finding the correct annealing temperature ideally with gradient pcr
Oof, that's a tough one. You could design one set of primers that amplifies BOTH genes (primer pair 1) and a second set of primers that is specific to the unique region in one gene (primer pair 2). Primer pair 2 will tell the expression of the paralog that has the unique region. The difference in amplification between the primer sets will tell you the expression of the gene without the unique region.
This is similar to what folks do to measure the relative abundance of mitochondrial DNA in when there is a mixed population of mtDNA that is full-length vs containing a deletion.
Also, have you looked at the 5' and 3' UTR regions? Those are transcribed into mRNA and typically have more variation than exons.
Paralog genes as such with my experience l did not find specific unique regions, but the amino acid alignments did show some few changes in amino acids at the 5' region and accordingly primer design was done. So even 3 amino acid changes is sufficient. It would be ideal if you have the whole CDs available.
Thank you all for important suggestions, will try to implement the given suggestions 😊
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