I am designing primers for RT qPCR for several genes. To check whether the primers bind to gDNA or not, I copy and paste forward and reverse primer sequences in Primer BLAST and I choose Refseq representative genome as Database. For some genes, the primer pair bind completely to "Primary Assembly" with same product length as mRNA for same gene, even though I have tried different sequence of interested mRNA.
The picture below is an example of primer for FGF23 mRNA which binds to primary assembly.
Does this mean the primer pair can bind to gDNA and it is not specific to my mRNA (cDNA) sequence?
Hi Tavana,
- For blasting I use Blastn, with "Human genomic + transcript", if human of course. It gives you both mRNA and gDNA bindings at the same time.
- As for designing primers that will selectively amplify mRNA and not your gDNA, you shall design them in separate exons. Therefore you will have a lenght shift and, if any gDNA amplification occurs, you will detect it with your melting curve.
- you can find exon borders on UCSC Genome Browser, but you might try as well some other softwares, like http://fasterdb.ens-lyon.fr/faster/home.pl
All the best,
Unless your primers span two exons with one primer, then they will always amplify gDNA along with cDNA. If your primers flank an intron sequence, then the gDNA product will be larger than the cDNA product. It's an easy way to check for gDNA contamination.
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