Real-time monitoring of DNA hybridization and replication using optical and acoustic biosensors Dissertation zur Erlangung des Grades
- „Doktor der Naturwissenschaften, Gudrun Stengel
Base pair stacking free energy parameters in a low ionic strength solvent were determined from an analysis of DNA fragments using temperature gradient gel electrophoresis (TGGE). Transition midpoint temperatures (Tu) were determined for the first melting domain (52 +/- 4 bp) of 16, 339 bp DNAs that differed from each other by single base pair substitutions. The data were combined with previously obtained Tu data from 17 similar DNAs that had single base pair changes at different sites [Ke, S. H., and Wartell, R. M. (1995) Biochemistry 34, 4593-4599]. The Tu values were used to evaluate free energy differences (deltaDeltaG) between 31 pairs of DNAs. Linear equations relating the deltaDeltaG values to changes in base pair stacking were analyzed by singular value decomposition (SVD) to determine the 10 nearest neighbor free energy parameters. The order of stability of the parameters, TA < AT < AA < AG < GT approximately TC approximately TG < CC < GC approximately CG, was essentially the same as the hierarchy determined in 1 M Na+ [Allawi, H. T., and SantaLucia, J., Jr. (1997) Biochemistry 36, 10581-10594]. The experimental free energy differences were in good agreement with predictions made using nearest-neighbor parameters determined from several previous studies conducted in medium or high salt concentrations. Conversely the parameters determined in the current study produced good predictions of free energy differences previously determined from 59 DNA oligomers in 1 M Na+. The results indicate that differences between base pair stacking energies are conserved across a wide range of ionic conditions, and in both oligomer and polymer DNA contexts.