Primers - Primers (Oct/28/2010 )
I am going to see FRO1 (AF405422.2 gene expression in peas by qRT-PCR. I have designed primers using primer3plus software. How I can know that my primes are okay and do not form any intron binding? How Can I also know that primers I got are on the exon region of the gene? Thanks.
AF405422 is a mRNA sequence, so it doesn't contain introns only joined exons. Primer3 designs primers that should not have high self or dimer complementarity.
For specificity check I would use BLASTn on each of your primers (but I'm not sure how covered the Pisum sativum genome is in Entrez).
Normaly I design primers intron-spanning and check the possition with Ensembl, but it doesn't contain Pisum Sativum genebuild. Nor can I find Entrez Gene page for that gene of yours. Sometimes the accession sequence contains exon boundary information, but not in your case. So, I'm affraid I can't help you, maybe someone with more experience with plant genetics know where to find better database.
Thanks. Since AF405422.2 is mRNA sequence, there should be no intron as you said. So normally I can trust the primers that primer3 designed, isnt it? Yeah, pisum genome is not available in databases. That's a problem.