Question

What component of the nucleotide is responsible for the absorption of ultraviolet light? How is this technique important in the analysis of nucleic acids?

Short answer

Answer: The nitrogenous base present in the nucleotide is responsible for the absorption of ultraviolet light due to its conjugated double bonds. This property is crucial in nucleic acid analysis for applications such as quantification, purity assessment, and monitoring structural changes or damage.

Step-by-step solution

Identify the component of the nucleotide responsible for ultraviolet light absorption

The component responsible for the absorption of ultraviolet light is the nitrogenous base present in the nucleotide. Nucleotides are the building blocks of nucleic acids, including DNA and RNA. They are composed of three parts: a sugar molecule (deoxyribose in DNA and ribose in RNA), a phosphate group, and a nitrogenous base. There are four types of nitrogenous bases in DNA: adenine (A), guanine (G), cytosine (C), and thymine (T); and in RNA: adenine (A), guanine (G), cytosine (C), and uracil (U).

Explain the absorption of ultraviolet light by nitrogenous bases

Nitrogenous bases are made of aromatic rings containing conjugated double bonds that can absorb ultraviolet light. Absorption occurs because the electrons in these double bonds are excited when exposed to ultraviolet light, moving them from a lower energy state (ground state) to a higher energy state (excited state). This property allows nucleic acids containing these nitrogenous bases to absorb UV light, with maximum absorption occurring typically around a wavelength of 260 nm.

Discuss the importance of this technique in nucleic acid analysis

The ability of nucleic acids to absorb ultraviolet light provides a useful tool in their analysis. Some important applications of this property include: 1. Quantification of nucleic acids: By measuring the absorbance of a nucleic acid solution at 260 nm, researchers can determine its concentration. This is based on the Beer-Lambert Law, which relates the amount of absorbed light to the concentration of the absorbing species. It is essential for the accurate preparation of samples and normalization of results in various molecular biology experiments. 2. Purity assessment: The ratios of absorbance at 260 nm and 280 nm (A260/A280) are used to assess the purity of nucleic acid samples. Proteins, which also absorb UV light, have maximum absorbance around 280 nm. A pure DNA sample will have an A260/A280 ratio of approximately 1.8, while a pure RNA sample will have a ratio of approximately 2.0. Lower ratios indicate contamination with proteins or other interfering substances. 3. Detection of structural changes and damage: Ultraviolet absorption can be employed to monitor changes to the DNA or RNA structure, such as the formation of secondary structures or damage induced by exposure to UV radiation, chemicals, or other factors. This is the basis for techniques such as UV melting analysis, which measures the change in absorbance as a function of temperature to assess nucleic acid stability and structural transitions. In conclusion, the nitrogenous bases in nucleotides are responsible for the absorption of ultraviolet light due to their conjugated double bonds. This property is crucial in nucleic acid analysis in applications such as quantification, purity assessment, and monitoring structural changes or damage.