intron/exon spanning primers - (Apr/26/2010 )
Since RNA yield from serum is always very low, I want to avoid treating my seru, samples with dnase after extracting the RNA. In understand to be able to avoid this step, my primer whousl be intro-exon spanning. Do you know of any website which can check if my existing primers are intro/exon spanning? I know there are websites which privide designers taking the intro/exon spanning but the website that I used (primer 3) for selecting my primers, didn't have that option and I just want to confirm.
Just remember that not only the assay has to be intron spaning to avoid amplifying DNA, but also the spanned intron must be long enough not to amplify fully. The best way to do it is to have the primer itself designed on the exon-exon boundary so the DNA wouldn't amplify at all.
You can check your primers on Ensembl, find your gene, select your transcript and select cDNA for display, then search in page for your primers. Ensembl shows exons in different colors so you can see if the primers are intron spanning.
There is a way how to design intron spanning primers in primer3, you just mark the exon-exon junction as a target and let the aplication design the primers. But this will design usualy primers on different exon (i.e. not the primer itself on the exon-exon junction), which is not that good. Primer Blast uses primer3 algorithm to design primers and has the option to design primers on the exon-exon junction.