We have designed RT-PCR Primers based on CDSs predicted from NGS data with confirmed protein similarity of various NCBI protein data bases. Our predicted CDSs are not less than 100 AA. However, when we searched same mRNA from respective gene of arabidopsis, we found multiple CDSs (of lesser size of AA) plotting at different places of the mRNA with many gaps observed in our complete CDSs.
How to design the primer? based on complete cds (though having gaps in comparison to the mRNA and we already tried and failed to get amplification) or based on CDS aligned with no gaps on mRNA ??
As you already said, your CDS are predictions, not confirmed. Arabidoposis has a LOT of duplicated genes and large gene families so you normally see similar sequences in several places in the genome. I would use the mRNA that is most similar to your CDS of interest as a starting point. You can test your primers on genomic DNA first (just know that the product sizes might be larger). If they amplify a product from gDNA, then test them on cDNA. This is going to be a very involved project with a LOT of trouble-shooting. Good luck!
As you already said, your CDS are predictions, not confirmed. Arabidoposis has a LOT of duplicated genes and large gene families so you normally see similar sequences in several places in the genome. I would use the mRNA that is most similar to your CDS of interest as a starting point. You can test your primers on genomic DNA first (just know that the product sizes might be larger). If they amplify a product from gDNA, then test them on cDNA. This is going to be a very involved project with a LOT of trouble-shooting. Good luck!
- Badges
- Science method