Studies of DNA Hybridization Reactions and Applications in Genetic Assays

The intent of this study was to investigate two fundamental aspects of short DNA duplex stability and how that stability differs for duplex molecules consisting of either all perfect match Watson/Crick base pairs or a mixture of perfect match Watson/Crick base pairs and mismatch base pairs. Theoretical and experimental investigations of the origins of the nucleation term in the free energy of DNA duplex formation were revisited. Thermodynamic parameters (ΔG, ΔH, Δ S and Tm) of short DNA/DNA duplexes ranging in length from 6 to 35 base pairs were systematically evaluated by Differential Scanning Calorimetry (DSC) as a function of sodium ion concentration. Extrapolation of the ΔG versus N plot to zero base pairs gave an estimate of the reference state free energy of a \u27hypothetical duplex\u27 having no hydrogen bonds, but still occupying precisely the same molar volume as the fully base paired duplex. This analysis provided a fresh evaluation of the free energy of duplex nucleation and new insights into the process of strand annealing. The second part involved quantitative evaluation of the influence of tandem mismatch base pairs on short DNA duplex stability as a function of Na+. For this investigation, DSC melting data were collected for 25 short duplex DNA molecules having increasing numbers of mismatches in two different topologies, i.e. on the \u27end\u27 or \u27interspersed\u27 in the duplex among Watson/Crick base pairs. Results revealed there is a definite influence of the position of mismatches on short duplex DNA stability. This analysis provides evaluation of the perturbation factors of the free energy associated with multiple mismatches in different topologies. Results achieved in the two phases of the work were combined to provide a new approach of calculating the thermodynamic stability of short DNA duplexes containing multiple mismatches. Tests of the new method to predict the free energies of independently characterized duplexes containing mismatch base pairs demonstrate that the model can be used to predict DNA stability