Multiple bands on gel or multiple peaks in the melting curve

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.

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