Clinical Chemistry · 2013 · 198 citations · 9 references
Genome‑sequencing has uncovered a vast number of SNPs, making primer design that avoids mismatches increasingly difficult. The study aimed to quantify how mismatches in primer‑annealing sites affect qPCR cycle thresholds, efficiency, and reproducibility. Using synthetic templates and primers, the authors tested mismatches across five commercial master mixes to assess qPCR performance. They found that mismatches’ impact grows with their number and proximity to the 3′ primer end, with ≥4 mismatches (or 3+2 across primers) abolishing amplification, while single mismatches >5 bp from the 3′ end have only moderate, concentration‑independent effects, guiding more reliable primer design in SNP‑rich regions.
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.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.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.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.
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Detection of PCR products using self-probing amplicons and fluorescence
David Whitcombe, Jane Theaker, Simon P. Guy et al. · Nature Biotechnology · 1999 · 696 citations