The thermodynamics of destabilizing DNA motifs such as bulges and mismatches are poorly characterized by melt curve analyses. I developed a new accurate and high- throughput method for measuring DNA motif thermodynamics, which I call TEEM. An experimental survey of biologically and biotechnologically relevant DNA motifs using TEEM revealed that the thermodynamic penalty of duplex destabilizing motifs are almost always temperature invariant, implying a dominant role of enthalpy. This phenomenon was remarkably general across disparate motifs such as bulges, mismatches, methylations, deaminations, and phosphorothioate backbone modifications, and directly contradicts prior DNA biophysical and biochemical models. To demonstrate the improved accuracy of structure prediction using the new thermodynamics parameters, I developed a new chemical probing method based on low-yield bisulfite conversion of cytosines. TEEM was also used to measure the stability of bulges at mononucleotide microsatellites which are clinically and forensically crucial DNA sequences. With this data and the partition function, I constructed a predictive model of the sliding bulge thermodynamics.