Exon-Exon Junction ? - (Apr/20/2009 )
Introductory question here:
I'm designing a whole list of primers to amplify cDNA for qPCR. I'm confused as to the idea of designing across an exon-exon junction to eliminate gDNA amplification. Does this mean that your actual primer sequence needs to span the exon-exon junction, or does it mean that the product created from the forward and reverse primers needs to span the exon-exon junction? If it's the former, (for example) I'm having trouble finding a forward primer sequence that fulfills all the needs of a good primer (i.e. 50%GC content, 80-150 product size, Tm~60degrees, no secondary structures, no primer dimers, 24 nucleotide primer sequence). I think I am misunderstanding the main idea here.
Any help is appreciated!
both is possible, just make sure that if the product spans the exon/exon junction that the intron between is big enough that it cant be amplified in PCR during elongation. eg. a 100bp intron would be coamplified, a 10kb intron rather not. you can also use this to see if you have gDNA in your RNA preparation. design primers for a HKG on different exons with a short intron in between, do conventional PCR and look on agarose gel if you also get the higher band.
Lynn on Apr 20 2009, 05:14 PM said:
I'm designing a whole list of primers to amplify cDNA for qPCR. I'm confused as to the idea of designing across an exon-exon junction to eliminate gDNA amplification. Does this mean that your actual primer sequence needs to span the exon-exon junction, or does it mean that the product created from the forward and reverse primers needs to span the exon-exon junction? If it's the former, (for example) I'm having trouble finding a forward primer sequence that fulfills all the needs of a good primer (i.e. 50%GC content, 80-150 product size, Tm~60degrees, no secondary structures, no primer dimers, 24 nucleotide primer sequence). I think I am misunderstanding the main idea here.I think it usually mean former, that primer should position between 2 exons.
However, one of primer cover exon junction should be sufficient to prevent gDNA amplify.
As for my previous work, it is very difficult to design primers cover exon junction at both end of amplification fragment. Thus, the way I design primer is: at least one primer cover exon junction and the amplification product cover a large intron, as tea-test suggest. And so far so good
Since the gDNA will still have the introns, while the cDNA does not, designing primers that will span exon-exon junctions ensures that the primers cannot associate with gDNA since the primers will have a sequence that does not exist in the gDNA. Even if they are still able to weakly associate with the gDNA, the amplicon size would be completely different between the cDNA and gDNA products due to the presence of the intron in the gDNA.
Example:
Primer 1 designed against exon 6 and exon 7 junction of gene X
Exon6-Exon7
cDNA for gene X
Exon1-Exon2-Exon3-Exon4-Exon5-Exon6-Exon7-Exon8
gDNA for gene X
Exon1-Intron-Exon2, etc. Exon6-Intron-Exon7-Intron-Exon8