SINGULAR-VALUE DECOMPOSITION OF 3-D DNA MELTING CURVES REVEALS COMPLEXITY IN THE MELTING PROCESS

The thermal denaturation of synthetic deoxypolynucleotides of defined sequence was studied by a three dimensional melting technique in which complete UV absorbance spectra were recorded as a function of tempera ture. The results of such an experiment defined a surface bounded by a bsorbance, wavelength, and temperature. A matrix of the experimental d ata was built, and analyzed by the method of singular value decomposit ion (SVD). SVD provides a rigorous, model-free analytical tool for eva luating the number of significant spectral species required to account for the changes in UV absorbance accompanying the duplex - to - singl e strand transition. For all of the polynucleotides studied (Poly dA - Poly dT; [Poly (dAdT)](2); Poly dG - Poly dC; [Poly(dGdC)](2)), SVD i ndicated the existence of at least 4-5 significant spectral species. T he DNA melting transition for even these simple repeating sequences ca nnot, therefore, be a simple two-state process. The basis spectra obta ined by SVD analysis were found to be unique for each polynucleotide s tudied. Differential scanning calorimetry was used to obtain model fre e estimates for the enthalpy of melting for the polynucleotides studie d, with results in good agreement with previously published values.