22.6. Thermal Stability
The temperature at which 50% DNA in given sample become denatured, known as melting temperature (Tm). The double stranded DNA show the less absorbance at 260 nm. While the denature DNA show the more absorbance at 260 nm this phenomena known as hyperchromatic effect.
Factors affecting Tm
- Ionic strength : When the lower the ionic strength is low it can not completely shield the anionic phosphate. So repulsion makes it energetically more favorable to separate the strands thus Tm temperature become lower.
- AT and GC Content : GC content has higher number of Hydrogen bond and base stacking interaction compare to AT content. So the high GC content has higher Tm than DNA with high AT content.
- pH : If the pH is greater than 10, the hydrogen bonds of Nitrogenous bases are destroyed and DNA become denatured. If the pH is less than 3, it also disrupt, the base pairing due to extensive protonation. So very high or very low pH lowers the Tm.