As my RNA sequence will only span exons and my primers will span exon-exon junctions will I get an amplification if I use genomic DNA in the qPCR with these primers? If not can someone please suggest whats the best gthing to do under such circumstances?
Many Thanks
Be aware of the pseudogenes - if your primers have homology with a pseudogene corresponding to your gene-of-interest, their annealing may result in undesired amplification products
Dear Sadia, I feel it would depend on the gene structure and how many introns span your sequence.
1. If you design primers with sequences from the 1st and last exons (transcription start site and stop site) then depending on your extension time and the length in between you may get the amplicon.
2. If you design a primer spanning the exonic junctions you may amplify the exons separately.
You will probably get an amplicon since SYBR Green mix contains a normal Taq DNA polymerase. The problem is the intent of your experiment. If you wish to quantify gDNA by qPCR using this sort of primers you may end with a large amplicon which will decrease qPCR efficiency and mislead your quantification. I've done gDNA quantification using qPCR but my amplicon was 110bp. You should check your primers in the gDNA context to verify the expected size. If your primers bind the end of one exon and the beginning of the following exon, and the intron isn't very long it MIGHT work.
Thanks you everyone for your answers. Andre could you please let me know how can i check for check primers in the gDNA context to verify the expected size?
Cheers,
Sadia
Hi, Sadia,
You can check your primers "in silico" using a sequence analyser software. If you can download the gDNA sequence of your organism from NCBI, then you would be able to test your primers with it and estimate the size of your amplicons.
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