DENATURATION

The two strands of DNA helix are held together by hydrogen bonds (change in pH or increase in temperature) results in the separation of polynucleotide strands. This phenomenon of loss of helical structure of DNA is known as denaturation. The phosphodiester bonds are not broken by denaturation. Loss of helical structure can be measured by increase in absorbance at 260 nm. The transition from native to a denatured form is usually very abrupt and is accelerated by reagents like urea and formamide, which enhance the aqueous solubility of the purine and pyrimidine groups. Denaturation involves the following changes:

Increase in absorption of ultraviolet light (= hyperchromic effect). All nucleic acids show a strong absorption in the ultraviolet with a maximum near 260 mμ. When the native DNA is denatured, there occurs a marked hyperchromicity, that is, an increase in the optical absorbency of the purine and pyrimidine bases. This change reflects a decrease in hydrogen bonding.

Decrease in specific optical rotation. Native DNA shows positive rotation, which is decreased upon denaturation. This change is analogous to the change observed in protein denaturation.

Decrease in viscosity. Due to the rigid double helical structure and rod-like character of DNA, the native DNA possesses high viscosity.