Polynucleotides melting curves

Whereas proteins have their low energy absorption band at 280 nm, polynucleotides typically have maxima at 260 nm (38,500 cm ). A phenomenon of particular importance in the study of nucleic acids is the hypochromic effect. In a denatured polynucleotide the absorption is approximately the sum of that of the individual components. However, when a double helical structure is formed and the bases are stacked together, there is as much as a 34% depression in the absorbance at 260 nm. This provides the basis for optical measurement of DNA melting curves (Fig. 5-45).45,86 The physical basis for the hypochromic effect is found in dipole-dipole interactions between the closely stacked base pairs.7,86,87... 

Figure 40. Mixing-, melting-curves and ORD-spectra of polynucleotide/polymer interactions (varied nucleohase design), c = lO M (sum of bases), pH 7.2/PBS...

E. G. Richards On the analysis of melting curves of stacked polynucleotides ... 

Like double-stranded DNA, synthetic polynucleotide complexes containing complementary bases show cooperative melting curves, i.e., the property studied (UV absorption, optical activity, viscosity, etc.) shows a sharp transition at a specific temperature. This Tm depends on ionic strength and, to a certain degree, on pH. If the temperature is increased above the Tm, however, a further continuous variation in the property studied will be observed. It is only in recent years that an understanding of such noncooperative phenomena is emerging. Here the studies of oligomers by CD and NMR were of critical importance. 

Fig. 7. Typical thermal denaturation curve plotting the uv absorbance at 260 nm (A260) of a polynucleotide sample versus temperature. The melting temperature (Tm) of this sample is ca 48° C.

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