Primer-Template Mismatch Does Not Contribute to Irreproducibility of PCR Replicates

Primers incorporating a range of mutations and targeting an area of the lacZ gene were tested for variability among PCR replicates. The coefficient of variance obtained from 3 PCR replicates for each mutated primer was compared with that obtained using the non-mutated primer. A control amplicon was incorporated into each reaction; variability in the ratio of the control and test amplicon integrals was used as an indicator of replicate variance. Qualitative and quantitative analysis both indicated that primer-template mismatch does not contribute significantly to replicate variability. However, very few replicates were used in this study; a larger set should be tested to confirm these findings.