qPCR Primer Design and Genomic DNA Amplification - (Aug/31/2004 )
I am having a problem designing qPCR primers that don't amplify genomic DNA. I design my primers in the appropriate manner (primers span exon-exon boundary and/or a large intron between the two primer pairs). When I run the PCR on a gel I see one band of the expected size. However, when I run the realtime PCR (on an ABI 7900HT) I see amplification in both -RT and genomic DNA controls. When I look at the dissociation curve I see only one large peak for each reaction. Is it possible that genomic DNA is being amplified or is this probably due to contamination. Help!!!
Thanks in advance. Nigel
If you have been running the same amplicon for some time, contamination is a very likely cause. You might consider using UNG or try replacing all reagents and see if it helps. How about the NTC?
Still, have you tested whether any pseudogenes are reported for your particular gene? You might add in a genomic control reaction, which has one primer in an intron as a "positive" DNA control. If this one does not show up in the -RT, but your problem primer still does, you might have a pseudogene problem.
Søren M. Echwald, MSc., Ph.D.
One Real-time PCR kit, which covers 38.565 genes