Question

What is the value of T_m (melting temperature) dependent on? Include a typical graph for DNA thermal denaturation and explain it qualitatively. Propose a method using UV spectroscopy to experimentally determine the melting temperature for DNA chains.

Answer 1

Ans: DNA is a double helical structure consisting of a sense and antisense strand. The two strands of DNA are complimentary to each other with four nitrogenous bases G (guanine) and C (cytosine), and A (adenine) and T (thymine) that are joined by hydrogen bond. With the rise in temperature hydrogen bonds are broken and unfolding of the strands take place forming two single strands of DNA. This process is called as melting.

Melting temperature, Tm shows how DNA strand will be hybridized. Tm depends on the base composition or the oligonucleotides concentration i.e., high CG increases Tm. It also depends on the DNA length. Longer DNA strand has higher Tm. It also depends on the concentration of ions (ionic strength) and compounds or the pH of the solution i.e., high salts in the solution increases the Tm.

DNA shows an UV absorption peak at 260 nm and absorbance increases with the DNA melting. This is due to the heterolytic rings present in the nucleotides.

With rise in temperature, AT rich region of the DNA start melting and leads to an increased absorbance. With further rise in temperature there is a sharp rise in the absorbance and the DNA gets completely denatured at higher temperature.

Method using UV spectroscopy to experimentally determine the melting temperature for DNA chains: Turn on the UV spectrophotometer and set absorbance to 260 nm and temperature to 20°C. Then, measure the absorbance of the DNA sample at 260 nm against the buffer which is used for dissolving the DNA. Absorbance should be in-between (0.1 – 0.4) for detecting the melting temperature. If absorbance is greater than 0.4, then dilute the sample in the buffer to get 1ml DNA solution with absorbance in-between (0.2 – 0.3). Now measure the absorbance of diluted DNA sample at 25°C. After that increase the temperature by 5°C and measure the absorbance till the cell attains the temperature. Repeat the step till the temperature reaches to 95°C. Record the measurements in the table.