A long term goal of the proposal is to obtain an accurate thermodynamic description of the interactions which govern DNA base pair opening. Interaction parameters will be empirically evaluated by comparing experimental denaturation curves of selected DNA molecules with theoretical predictions. UV absorbance and polyacrylamide gel electrophoresis will be used to monitor DNA duplex-coil transitions. Three aspects of DNA stability will be examined; stacking heterogeneity, the formation of cruciform and/or hairpin structures in linear DNA, and electrostatic end- effects. Base pair stacking interactions will be evaluated in solvents containing 0.1 M to 0.5 M Na+. Two differential melting curves from DNA oligomer.
A second aim i s to understand how certain inverted repeat sequences influence DNA melting. Theoretical and experimental studies will be made to evaluate the factors governing the formation of cruciform structures during the melting of linear DNAs. Preliminary studies indicate that certain inverted repeat sequences can form hairpin-like structures during the melting of linear DNAs. Electrostatic end-effects will be evaluated from melting data of a set of DNA oligomer differing in length. A second goal of this application is to apply a temperature gradient electrophoresis procedure to studies on DNA melting. An apparatus has been constructed which simplifies features of denaturing gradient gel electrophoresis. It will be employed to examine the effect of single base pair changes on DNA stability. Predicted DNA melting behavior will be compared to temperature induced melting in a gel environment. Eight DNA fragments differing by single base pair changes will be studied. A method for altering the thermal stability distribution along a DNA will also be examined. Improved understanding of DNA melting in a gel environment, and the development of methods to localize base pair changes may enhance the detection of genetic polymorphisms and mutations.
