Entering edit mode
5.0 years ago
l.souza
▴
80
Hello everyone,
I have an exogenous gene inserted in a plant to quantify via RT-qPCR. However, the transformed plant also has an endoogenous copy of this gene. Is there a way to design a pair of primers for the exogenous gene without binding to the endogenous one? At Primer-BLAST there's an option to specify organisms, but I don't know any option to do the opposite.
Not really bioinformatics so your question might get closed. Anyway, is the inserted gene a full-length gene body with exons and introns or just the CDS? Is it 100% identical with the endogenous one?
Tools for primer design is not regarding Bioinformatics? The inserted gene is just the CDS... It is not 100 % identical with the endogenous one; however, all primers I've designed with Primer3 also hit in the endogenous one.
It is borderline but mainly a wet-lab problem. Anyway, is the insertion site known for that gene? If so I would try to force primer3 to select a primer spanning part of the genomic up/downstream region and the inserted CDS. Also be sure to have control primers against genomic DNA to check for gDNA contaminations and make sure you properly treat your RNA with DNase. To distinguish endo- and exogenous gene, maybe use a primer that binds to the UTRs of the native one.
Are upstreams and downstreams the same for both gene copies, or it is not known?
If they are very close in sequence there is going to be very little you can do to ensure binding only to the exogenous copy.
Your best bet may be to design flanking primers if the region just outside the gene is different.