Background: Robust designs of PCR-based molecular diagnostic assays rely on the discrimination potential of sequence variants affecting primer-to-template annealing. However, for accurate quantitative PCR (qPCR) assessment of gene expression in populations with gene polymorphisms, the effects of sequence variants within primer binding sites must be minimized. This dichotomy in PCR applications prompted us to design experiments to specifically address the quantitative nature of PCR amplifications with oligonucleotides containing mismatches.
Results: We performed qPCR reactions with several primer-target combinations and calculated ratios of molecules obtained with mismatch oligonucleotides to the average obtained with perfect match primer pairs. Amplifications were performed with genomic DNA and complementary DNA samples from different genotypes to validate the findings obtained with plasmid DNA. Our results demonstrate that PCR amplifications are driven by probabilities of oligonucleotides annealing to target sequences. Empiric probabilities can be measured for any primer pair. Alternatively, for primers containing mismatches, probabilities can be measured for individual primers and calculated for primer pairs.
Conclusion: The ability to evaluate priming (and mispriming) rates and to predict their impacts provided a precise and quantitative description of assay performance. Priming probabilities were also found to be a good measure of analytical specificity.