BACKGROUND: Genome-sequencing studies have led to an immense increase in the number of known singlenucleotide 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

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... ct 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 3Ј end of the primer. In addition, having Ն4 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 Ͼ5 bp from the 3Ј 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 Ն4 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. 3 Nonstandard abbreviations: qPCR, quantitative PCR; SNP, single-nucleotide polymorphism; Cq, quantification cycle; dCq, difference between the Cq for the mismatch reaction and the mean Cq of the corresponding perfect-match reactions; dT m , difference in primer annealing temperature; T m , primer annealing temperature.