Thermal Melting of Duplex DNA in the Slow Exchange Regime

Abstract

The thermal melting of small DNA duplexes usually occurs as a two-state transition in which the duplex and its component single strands are co-populated throughout the transition. Since a duplex and its single-stranded forms can be resolved by free solution capillary electrophoresis, this technique can be used to monitor the kinetics of denaturation. When a duplex and its component single-strands are in fast exchange, a single peak is observed in the electropherograms, with a mobility that is proportional to the weighted average of the mobilities of the duplex and its component single strands at various temperatures. However, if melting occurs in the slow exchange regime, resolved peaks are observed for the duplex and its component single strands. The area under the peak corresponding to the duplex progressively diminishes with increasing temperature, while the area under the peak(s) characteristic of the single strands progressively increases. Although the melting of small DNA hairpins and duplexes often occurs in the fast exchange regime, the melting of DNA duplexes containing internal loops occurs in the slow exchange regime. The equilibrium and kinetic parameters accompanying the melting of DNA duplexes with internal loops will be reported.