Hi all,
I am going to perform some RT-qPCR tests to validate an experiment.
I'm currently in the process of ordering primers, and I would like to get them from the Harvard Primer Bank since these have been validated before.
However, I would like to get primer pairs that span an exon junction (i.e., one primer on one exon, the other primer on another exon.)
Currently, I have to do a lot of steps to figure this out, since the Primer Bank only gives the positions of the primers on the CDS, and not on the entire mRNA molecule.
Does anyone have a quick way to check on which exons the primers belong?
Thank you!
Hi Gael
PRIMER BLAST is part of the Genbank suite of tools and will target primers to exon junctions for real time PCR if you specify that as a requirement; it is also not hard to locate the position of such primers in the context of its CDS sequences
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Hi Gael
PRIMER BLAST is part of the Genbank suite of tools and will target primers to exon junctions for real time PCR if you specify that as a requirement; it is also not hard to locate the position of such primers in the context of its CDS sequences
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
There is no software to that, another way is to first do pairwise alignment with your genomic sequence and CDS sequence it will help you to understand and see exons (where alignments matches from genomic and CDS sequence) and introns (where only genomic seq is present in alignment). Later search your primer sequence using Ctrl+F key on the alignmnet and you can easily find out where primer sits in the sequence. You can do pairwise alignment using http://www.ebi.ac.uk/Tools/psa/ . Hope it helps.
Hi again
to reiterate: software tools like primer BLAST and the real
time primer design tool from Roche will target exon exon boundaries; that is the essence of real time primer design and hence tools which perform this function
primer BLAST when performing this role utilises its own gene bank CDS sequences and hence it is relatively straightforward to locate the exact position of intron spanning/exon junction primers in the context of those sequences
Thanks Laurence I was not aware of that. I use to design exon spanning primers by PRIMER BLAST by setting the region where I want to design the primers. I learned something new. Thanks once again.
Yes.
Check UCSC Genome Browser, excellent site. Under this go to BLAT.
There is a program called perprimer http://perlprimer.sourceforge.net/tutorial.html that allows you to see and design primers spanning exon bounderies. You do so by inserting both the genomic and cDNA sequence of the gene you're targeting and then blast that with whatever parameters you want. Easy to use.
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