Single-nucleotide polymorphisms and other mismatches reduce performance of quantitative PCR assays.

Background: Genome-sequencing studies have led to an immense increase in the number of known single-nucleotide polymorphisms (SNPs). Designing primers that anneal to regions devoid of SNPs has therefore become challenging. We studied the impact of one or more mismatches in primer-annealing sites on different quantitative PCR (qPCR)-related parameters, such as quantitative cycle (Cq), amplification efficiency, and reproducibility.

Methods: We used synthetic templates and primers to assess the effect of mismatches at primer-annealing sites on qPCR assay performance. Reactions were performed with 5 commercially available master mixes. We studied the effects of the number, type, and position of priming mismatches on Cq value, PCR efficiency, reproducibility, and yield.

Results: The impact of mismatches was most pronounced for the number of mismatched nucleotides and for their distance from the 3' end of the primer. In addition, having ≥4 mismatches in a single primer or having 3 mismatches in one primer and 2 in the other was required to block a reaction completely. Finally, the degree of the mismatch effect was concentration independent for single mismatches, whereas concentration independence failed at higher template concentrations as the number of mismatches increased.

Conclusions: Single mismatches located >5 bp from the 3' end have a moderate effect on qPCR amplification and can be tolerated. This finding, together with the concentration independence for single mismatches and the complete blocking of the PCR reaction for ≥4 mismatches, can help to chart mismatch behavior in qPCR reactions and increase the rate of successful primer design for sequences with a high SNP density or for homologous regions of sequence.