Denaturation of DNA

Denaturation of DNA

We have already seen that the hydrogen bonds between base pairs are an important factor in holding the double helix together. The amount of stabilizing energy associated with the hydrogen bonds is not great, but the hydrogen bonds hold the two polynucleotide chains in the proper alignment. However, the stacking of the bases in the native conformation of DNA contributes the largest part of the stabilization energy. Energy must be added to a sample of DNA to break the hydrogen bonds and to disrupt the stacking interactions. This is usually carried out by heating the DNA in solution.

How can we monitor DNA denaturation?

The heat denaturation of DNA, also called melting, can be monitored experimentally by observing the absorption of ultraviolet light. The bases absorb light in the 260-nm-wavelength region. As the DNA is heated and the strands separate, the wavelength of absorption does not change, but the amount of light absorbed increases (Figure 9.18). This effect is called hyperchromicity. It is based on the fact that the bases, which are stacked on top of one another in native DNA, become unstacked as the DNA is denatured.

Because the bases interact differently in the stacked and unstacked orienta-tions, their absorbance changes. Heat denaturation is a way to obtain single-stranded DNA (Figure 9.19), which has many uses. When DNA is replicated, it first becomes single-stranded so that the complementary bases can be aligned. This same principle is seen during a chemical reaction used to determine the DNA sequence . A most ambitious example of this reaction is described in the following Biochemical Connections box.

Under a given set of conditions, there is a characteristic midpoint of the melting curve (the transition temperature, or melting temperature, written T_m) for DNA from each distinct source. The underlying reason for this prop-erty is that each type of DNA has a given, well-defined base composition. A G–C base pair has three hydrogen bonds, and an A–T base pair has only two. The higher the percentage of G–C base pairs, the higher the melting temperature of a DNA molecule. In addition to the effect of the base pairs, G–C pairs are more hydrophobic than A–T pairs, so they stack better, which also affects the melting curve.

Renaturation of denatured DNA is possible on slow cooling (Figure 9.18). The separated strands can recombine and form the same base pairs responsible for maintaining the double helix.

Summary

The two strands of the double helix can be separated by heating DNA samples. This process is called denaturation.

DNA denaturation can be monitored by observing the rise in ultraviolet absorption than accompanies the process.

The temperature at which DNA becomes denatured by heat depends on its base composition; higher temperatures are needed to denature DNA rich in G–C base pairs.