Question

What is the hyperchromic effect? How is it measured? What does \(T_{m}\) imply?

Short answer

Answer: The hyperchromic effect is the increase in absorbance of UV light by DNA or RNA molecules as they undergo denaturation, leading to the separation of the double helix strands. It can be measured using a spectrophotometer. The melting temperature (\(T_{m}\)) is the temperature at which half of the DNA or RNA molecules are denatured, indicating the stability of the double helix. The \(T_{m}\) can be determined from the temperature-absorbance curve obtained during the measurement of the hyperchromic effect.

Step-by-step solution

Definition of the Hyperchromic Effect

The hyperchromic effect refers to the increase in the absorbance of ultraviolet (UV) light by DNA or RNA molecules as they undergo denaturation. Denaturation is a process in which the hydrogen bonds holding together the nucleotide base pairs in a double helix are broken, causing the two strands of DNA or RNA to separate. This separation leads to the increased exposure of the nitrogenous bases, which results in greater absorbance of UV light (primarily at 260 nm wavelength).

Measuring the Hyperchromic Effect

The hyperchromic effect can be measured using a spectrophotometer, an instrument that measures the amount of light absorbed by a sample at specific wavelengths. To measure the hyperchromic effect of a DNA or RNA sample, the absorbance of UV light (typically at 260 nm) is recorded as the temperature increases. As the temperature rises and the sample denatures, a clear increase in absorbance can be observed.

Understanding Melting Temperature (\(T_{m}\))

The melting temperature (\(T_{m}\)) is defined as the temperature at which half of the DNA or RNA molecules in a sample are denatured. In other words, it's the temperature at which 50% of the molecules have separated into single strands. A higher \(T_{m}\) indicates a stronger bond between the two strands of the double helix, usually due to a higher proportion of guanine-cytosine (GC) base pairs, which form three hydrogen bonds compared to the two hydrogen bonds formed by adenine-thymine (AT) base pairs. The \(T_{m}\) can be determined from the temperature-absorbance curve obtained during the measurement of the hyperchromic effect. In summary, the hyperchromic effect is the increase in absorbance of UV light by DNA or RNA molecules as they denature, and it can be measured using a spectrophotometer. The melting temperature (\(T_{m}\)) is an important concept in understanding the stability of the DNA or RNA double helix, as it represents the temperature at which half of the molecules have denatured.