Agarose gel electrophoresis or melting curve analysis may not always reliably measure PCR specificity. Based on our experience, bimodal melting curves are sometimes observed for long amplicons (>200 bp) even when the PCRs are specific. The observed multiple peaks may be the result of ampli-con sequence heterogeneity rather than non-specificity. On the other hand, for short amplicons (<150 bp) very weak and fussy bands migrating ahead of the major specific bands are sometimes observed on agarose gel. These weak bands are super-structured or single-stranded version of the specific amplicons in equilibrium state and therefore should be considered specific. Although gel electrophoresis or melting curve analysis alone may not be 100% reliable, the combination of both can always reveal PCR specificity in our experience.
Was this article helpful?