Question

The UV absorbance of a solution of a double-stranded DNA is monitored at 260 nm as a function of temperature. Data appear in the following table, where temperature (T) and absorbance (measured at 260 nm) are shown. From the data determine the melting temperature.

T(K): 343 348 353 355 357 359 361 365 370

Absorbance: 0.3 0.35 0.5 0.75 1.22 1.40 1.43 1.45 1.47

Note: Please remember that you use highly concentrated samples where absorbance can be as high as shown in this table. This will undermine the accuracy and potentially deviation from Beer’s law.

Answer 1

Hyperchromicity is the increase of absorbance (optical density) of a material. The most famous example is the hyperchromicity of DNA that occurs when the DNA duplex is denatured. The UV absorption is increased when the two single DNA strands are being separated, either by heat or by addition of denaturant or by increasing the pH level. The opposite, a decrease of absorbance is called hypochromicity.

Hyperchromicity can be used to track the condition of DNA as temperature changes. The transition/melting temperature (T_m) is the temperature where the absorbance of UV light is 50% between the maximum and minimum, i.e. where 50% of the DNA is denatured.

So, the graph for the given data is given below and Tm is found to be 355.30 (⁰C).