Single-Nucleotide Polymorphisms and Other Mismatches Reduce Performance of Quantitative PCR Assays

Abstract

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.

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

“…Ideally these primers have a SNP at their 3' end. Primers with less allele-specificity can be used if they display a minimum of a 5 Ct value difference in their amplification of the correct vs. the incorrect genotype 25 . Primers that carry a SNP more 5' than the 4 th base from the 3' end usually fail to exhibit allele specificity and amplify both genotypes with equal efficiency, revealing the importance of the SNP position in the primer 34 .…”

“…Primers that carry a SNP more 5' than the 4 th base from the 3' end usually fail to exhibit allele specificity and amplify both genotypes with equal efficiency, revealing the importance of the SNP position in the primer 34 . In addition a purine/purine or pyrimidine/pyrimidine substitution has been shown to have a greater impact on the T m difference of two primers compared to a purine/pyrimidine substitution 25 .…”

“…On average there is a SNP every 125 bp between these two genomes, providing flexibility in primer design for expression and genotyping analyses. The presence of one SNP can influence the primer melting temperature (T m ) and target specificity in real-time quantitative PCR (qPCR) amplification allowing for discrimination of the two alleles 25 . Furthermore a mismatch within the 3' end of the primer greatly influences the ability of DNA polymerase to extend from the primer preventing amplification of the undesired allele target 26 .…”

PCR bias associated with conserved primer binding sites, used to determine genotype diversity within Citrus tristeza virus populations

“…The Illumina MiSeq data corroborates the unexpectedly high representation of B165. The apparent lack of bias against this genotype may be due to the fact that only one of the eight nucleotide mismatches occurred within the critical 5 bp of the 3' end of these primers, while the others are likely to have only moderate effects on amplification efficiencies (Lefever et al, 2013;Waud et al, 2014). This was further supported by the data obtained to the F-region, where all of the forward primers, except that specific to T30, contained a nucleotide that differed from the consensus, 4 bp from the 3' end of each primer, probably resulting in the preferential amplification of T30.…”

“…Therefore, the single mismatch at the midpoint of the Taiwan-Pum/SP/T1 and RB forward primer binding sites could have been responsible for the reduction in amplification efficiencies of these templates. The generally accepted tolerances for primer mismatches during amplification (Lefever et al, 2013) are clearly complex and should be tested empirically.…”