qPCR cycling programs and protocols.

In quantitative polymerase chain reaction (qPCR) experiments, primers containing mismatches with respect to the template are widely used in measuring repetitive DNA elements. Primer-template mismatches may lead to underestimation of the input sample quantity due to inefficient annealing and amplification. But how primer-template mismatches affect quantification accuracy has not been rigorously investigated. In this study, we performed a series of qPCR experiments in which we tested three pairs of mismatched telomere primers (tel1/tel2, tel1b/tel2b and telg/telc) and two pairs of perfect-match reference gene primers (36B4-F/-R and IFNB1-F/-R) at three different primer concentrations under four cycling conditions. Templates used were genomic DNA from two human cell lines and oligo duplexes which contained telomere sequences, reference gene sequences, or both. We demonstrated that the underestimation of input sample quantity from reactions containing mismatched primers was not due to lower amplification efficiency (E), but due to ineffective usage of the input sample. We defined a novel concept of amplification efficacy (f) which quantifies the effectiveness of input sample amplification by primers. We have modified the conventional qPCR kinetic formula to include f, which corrects the effects of primer mismatches. We demonstrated that reactions containing mismatched telomere primer pairs had similar efficiency (E), but varying degrees of reduced efficacy (f) in comparison to those with the perfect-match gene primer pairs. Using the quantitative parameter f, underestimation of initial target by telomere primers can be adjusted to provide a more accurate measurement. Additionally, we found that the tel1b/tel2b primer set at concentration of 500 nM and 900 nM exhibited the best amplification efficacy f. This study provides a novel way to incorporate an evaluation of amplification efficacy into qPCR analysis. In turn, it improves mismatched primer selection and quantification accuracy in amplifying DNA repeats using qPCR methods.