For EGFR mutation analysis in Exon 18 to 21 by real time we use single tube having primers, WT probe tagged with VIC, Mutation probe tagged with FAM, Mastermix and 1 microlitre DNA (both WT and mutated). The DNA used is from formalin-fixed paraffin embedded tumor tissues. We are facing problems with WT amplifications in Exon 20. We think that the gap between primer and probe(150bp) is causing the problem in the amplification. While we are getting proper curves in all the other exons.
Personally, I would prefer verifying a new protocol using DNA extracted from fresh samples (knowing that formalin-fixation might result in DNA degradation). However, obtaining successful amplifications of the other exons indicates that your DNA might be intact.
Moreover, should I assume that both wildtype and mutant probes share the same annealing site? and how about the mutant probe? Is it working well? If yes, then it is not the gap; it should rather be related to the wildtype probe per se.
For troubleshooting, you might first check the primers (alone) using SYBR Green I (real-time PCR) or ethidium bromide (gel electrophoresis). If the amplification is successful, then check each probe separately. Optimizing the reaction conditions might help solve the problem. Hope this helps!
Personally, I would prefer verifying a new protocol using DNA extracted from fresh samples (knowing that formalin-fixation might result in DNA degradation). However, obtaining successful amplifications of the other exons indicates that your DNA might be intact.
Moreover, should I assume that both wildtype and mutant probes share the same annealing site? and how about the mutant probe? Is it working well? If yes, then it is not the gap; it should rather be related to the wildtype probe per se.
For troubleshooting, you might first check the primers (alone) using SYBR Green I (real-time PCR) or ethidium bromide (gel electrophoresis). If the amplification is successful, then check each probe separately. Optimizing the reaction conditions might help solve the problem. Hope this helps!
In addition to Wafa's comments above, it should also be noted that if the 150 bp gap only exists in the WT and not the [e.g., deletion?] mutant, such a sizeable gap between rev primer or fwd primer and probe can impact the efficacy of the PCR. Ordinarily you don't want sizeable gaps between adjacent oligo binding sites for good qPCR. If your DNA has indeed undergone enough degradation, sizeable gaps like this can be prone to inviting the lost portion into the assay - ending up with little or no amplification event. A picture of your set-up (primer/probe locations) would tell us more on this end.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques