Nucleic acid extraction is used in molecular biochemical tests and diagnosis, and is the first step of cloning, transformation, enzyme digestion, in vitro transcription, amplification, and sequencing. However, due to the large amount of proteins, carbohydrates, metabolites and other contaminants in the original sample, it is not easy to extract high-quality nucleic acids. It is necessary to apply the supporting nucleic acid extraction system and nucleic acid extraction reagents to automatically complete the extraction of sample nucleic acid.
Principles of nucleic acid extraction and purification
1. The integrity of the primary structure of nucleic acid should be guaranteed;
2. Eliminate pollution from other molecules.
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. The purification of nucleic acid should meet the following three requirements:
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;
2. 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 condition of pH 4-10;
3. Reduce the degradation of nucleic acids by physical factors. The main physical degradation factor is mechanical shearing force, 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 burst explosively, 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 shear force caused by water boiling, the high temperature itself can also damage 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.
4. To prevent the biodegradation of nucleic acids, 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, DNA enzymes require the activation of metal divalent ions Mg2 and Ca2. Using EDTA and citrate to chelate metal divalent ions can basically inhibit DNase activity. 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 material 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 high purity and high quality.