Impact of basepair mismatch in primer annealing on qPCR assay performance

Steve Lefever, Pattyn Filip, Speleman Frank, Hellemans Jan, Vandesompele Jo
Center for Medical Genetics Ghent, Ghent University, Belgium

With the onset of the 1000 Genomes Project that aims to explore human genetic variation, a multitude of new single nucleotide (SNP) polymorphisms have emerged. Therefore, the density and distribution of known variants has increased significantly and hampers design of primers in regions devoid of SNPs. Importantly, perfect primer/target complementarity is believed to be crucial in all samples since the presence of a SNP in the primer annealing region may affect the result of a quantitative polymerase chain reaction (qPCR). In the worst case, absence of amplification of the variant allele has been reported in literature. However, the impact of such a mismatch in relation to its location in the primer and local sequence context has not been systematically studied. To address this important issue, we have designed a study to systematically and empirically assess the relation between the presence of a mismatch in the last five 3’ end bases of a PCR primer and various qPCR parameters, such as efficiency and Cq-value. The study is based on a constant reverse primer and a set of nine walking forward primers, located up to four bases up- or downstream from a starting position, where single base pair mismatches have been introduced at the 3’ end final base. 36 forward primers and 16 synthetic oligonucleotide templates (good for 576 distinct and informative match/mismatch reactions) were designed to prevent secondary structure formation, self-annealing and primer dimerization. Different qPCR reaction mixes with variable sensitivity towards primer mismatches are evaluated. Data analysis is currently being finalized and results will be presented at the symposium.

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