Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves

摘要

Many factors that change the temperature position and interval of the DNA helix-coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, T-m, and temperature melting width, Delta T, of the entire DNA transition. Changes in T-m and Delta T can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining T-m and Delta T. Indeed, some of the methods give unreasonable "jagged" T-m and Delta T dependences on varying relative concentration of DNA chemical modifications (r(b)), [Na+], and GC content. At the same time, T-m determined as the helix-coil transition average temperature, and Delta T, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of T-m and Delta T on r(b), [Na+], and GC content. For multi-peak DMCs, T-m value determined in this way is the closest to the thermodynamic melting temperature (the helix-coil transition enthalpy/entropy ratio). (C) 2015 Published by Elsevier Inc.