DNA extraction principles

Nucleic acid includes two kinds of molecules, DNA and RNA, both of which exist in the state of being bound to proteins.

The general principles of nucleic acid separation and purification:

1. The integrity of the primary structure of the nucleic acid should be guaranteed; 2. The contamination of other molecules should be excluded.

In order to ensure the study of nucleic acid structure and function, a complete primary structure is the most basic requirement, because genetic information is all stored in the primary structure, and the primary structure of nucleic acid also determines the form of its high-level structure and other biological macromolecules. The way of combining. For the purification of nucleic acid, the following three requirements should be met:

1. There are no organic solvents and excessively high concentrations of metal ions that can inhibit enzymes in nucleic acid samples;

2. The pollution of other biological macromolecules such as proteins, polysaccharides and lipid molecules should be minimized;

3. Eliminate the contamination of other nucleic acid molecules, such as RNA should be removed when extracting DNA molecules, and vice versa.

In order to ensure the integrity and purity of the isolated nucleic acid, the following items should be noted during the experiment:

1. Try to simplify the operation steps and shorten the extraction process to reduce the damage of various harmful factors to the nucleic acid;

• To reduce the degradation of nucleic acids by chemical factors, in order to avoid damage to the phosphodiester bond in the nucleic acid chain by over acid and over base, the operation is mostly carried out under the conditions of pH 4-10;

• Reduce the degradation of nucleic acids by physical factors. The main physical degradation factor is mechanical shear, followed by high temperature. Mechanical shearing force includes strong and high-speed solution shaking and stirring to make the solution quickly pass through the narrow and long pores, the cells are suddenly placed in the hypotonic solution, the cells are explosively ruptured, and the DNA samples are repeatedly frozen and stored. These operating details should be paid attention to in the experimental operation. The main hazard of mechanical shearing is linear DNA molecules with large molecular weights, such as the chromosomal DNA of eukaryotic cells. The threat to circular DNA molecules with small molecular weights, such as plasmid DNA and RNA molecules, is relatively small. High temperature is like boiling for a long time. In addition to the shearing force caused by water boiling, the high temperature itself also has a destructive effect on some chemical bonds in nucleic acid molecules. In the process of nucleic acid extraction, it is generally operated at low temperature, but now it is found that there is not much difference in the quality of nucleic acid obtained at room temperature rapid extraction and low temperature extraction.

• To prevent the biodegradation of nucleic acid, various nucleases inside or outside the cell digest the phosphodiester bond in the nucleic acid chain and directly destroy the primary structure of the nucleic acid. Among them, DNase requires the activation of the metal divalent ions Mg2 and Ca2. Using EDTA and citrate to chelate metal divalent ions can basically inhibit DNase activity. The RNase is not only widely distributed, easily contaminates the sample, but also resistant to high temperature, acid, alkali, and not easy to inactivate, so it is the main hazard factor in the biodegradable RNA extraction process.

The main steps of nucleic acid extraction are nothing more than disrupting cells, removing proteins bound to nucleic acids, polysaccharides, lipids and other biological macromolecules, removing other unwanted nucleic acid molecules, precipitating nucleic acids, removing salts, organic solvents and other impurities, and purifying nucleic acids. Wait. The nucleic acid extraction scheme should be determined according to the characteristics of the specific biological materials and the nucleic acid molecules to be extracted. For the nucleic acid molecules enriched in a specific organelle, the scheme of extracting the organelle in advance and then extracting the target nucleic acid molecule can obtain completeness and Nucleic acid molecules with both high purity and high quality.

In the process of DNA extraction, we must abide by the principles. DNA Extractor is a fully automatic nucleic acid extraction system with a variety of models for you to choose from.