The Basics of Nucleic Acid Quantitation
Nucleic acid quantitation is an important process in molecular biology that aims to determine the average concentrations and purity of DNA and RNA present in a mixture to achieve an optimum performance.
Quantitation of nucleic acids can be done through spectrophotometric analysis and UV florescence in the presence of DNA dye.
Using a spectrophotometer, this method measures the concentration of nucleic acid in the solution based on how nucleic acids absorb the ultra-violet light in a specific pattern. The process involves exposing the sample to ultraviolet light at 260 nm, and a photo detector will measure the light that passes through the sample. The more light absorbed by the sample, the more nucleic acid is present in the sample.
Using the ratio of the absorbance at 260 nm and 280 nm, scientists also determine how contaminated the nucleic acids are, which will serve as the basis as to what DNA and RNA purification method they are to use. It is important to quantify the rate of nucleic acid contamination and purify them to avoid errors in DNA quantity estimation. For instance, if the nucleic acid is highly contaminated with phenol it may result to overestimation of DNA concentration.
Quantification using Florescent Dyes
This is an alternative method to measure the DNA in solutions or sample that have very low concentrations. In this method scientists use florescent dyes to measure the fluorescence intensity of dyes that bind to nucleic acids. Fluorescent dyes are colored substances that glow as they emit light during an electromagnetic radiation or when they are exposed to an ultraviolet light.
There are two approaches that scientists utilize in quantitation using fluorescent dyes. The first approach is using the spot test which involves placing the sample onto the agarose gel or plastic wrap. The scientist then adds the fluorescent dye to the agarose gel or to the samples in the plastic wrap and then exposes the sample to the ultraviolet light. Because of the fluorescent dye, the set of samples with known concentration will be spotted alongside the sample of unknown concentration.
The second approach is running the solution through polyacyramide gel. This process is also known as gel electrophresis. In this method, the sample is subject to an electric field to move the negatively charged molecules through an agarose matrix. Through the pores of the gel, the shorter molecules move faster together, while the longer molecules remain moving slow – allowing the proteins to separate from the solution.