(1) Release of nucleic acids
Under normal circumstances, both DNA and RNA are located in the cell, so the first step in nucleic acid isolation and purification is to break the cell and release the nucleic acid. There are many methods for cell disruption, including mechanical and non-mechanical methods. The mechanical method can be further divided into a liquid shearing method and a solid shearing method. The main hazard of mechanical shearing is high molecular weight linear DNA molecules, so this method is not suitable for the separation and purification of chromosomal DNA. Non-mechanical methods can be divided into drying methods and lysis methods. At present, most lysis methods are used. Among them, the lysis method using suitable chemical reagents and enzymatic lysing cells has high cleavage efficiency, mild method, high yield and good maintenance of nucleic acid integrity, and has been widely used.
(II) Isolation and purification of nucleic acids
Cell lysates are complex mixtures of nucleic acid-containing molecules that themselves may still bind to proteins. Under the premise of ensuring the integrity of nucleic acid molecules, it is not easy to separate a certain amount of nucleic acid molecules that meet the purity requirements. This requires us to fully understand the properties of nucleic acid molecules. An efficient scheme is designed to separate the nucleic acid from other substances in one or more properties. This difference is multifaceted, including differences in cell localization and tissue distribution, differences in physicochemical properties, and their unique biological properties. The contaminants that should be removed mainly include three parts: macromolecular contaminants that are not nucleic acids, undesired nucleic acid molecules, and solutions and reagents that are added to the subsequent experiments and applications during the separation and purification of nucleic acids. Non-nucleic acid macromolecular contaminants mainly include proteins, polysaccharides and lipids; non-required nucleic acid molecules refer to RNA as a contaminant when preparing DNA, and when DNA is prepared as a contaminant, when preparing a specific nucleic acid molecule Other nucleic acid molecules are contaminants; as for the organic solvent and some metal ions added during the separation and purification of nucleic acids, they often need to be well removed due to their influence on subsequent experiments.
(3) Relationship between nucleic acid quality and extraction steps
In general, the more separation and purification steps, the higher the purity of the nucleic acid, but the yield will gradually decrease and the integrity will be more difficult to ensure. On the contrary, by separating the experimental scheme with few purification steps, we can obtain more nucleic acid molecules with better integrity, but the purity is not necessarily high. This requires selection in conjunction with the use of the nucleic acid.
(4) Concentration, precipitation and washing of nucleic acids
With the gradual addition of nucleic acid extraction reagents and the inevitable loss of nucleic acid molecules during the removal of contaminants, the concentration of nucleic acids in the sample will gradually decrease, and will affect the subsequent experimental operations or meet the needs of subsequent research and application. The nucleic acid is concentrated. Precipitation is the most commonly used method for nucleic acid concentration. The advantage is that after the nucleic acid is precipitated, the lysis buffer can be easily changed and the nucleic acid solution can be adjusted to the desired concentration. In addition, the nucleic acid precipitate can also remove some impurities and certain salt ions. Purification effect. After adding a certain concentration of the salt, the nucleic acid is precipitated with an organic solvent. Among the commonly used salts are sodium acetate, potassium acetate, ammonium acetate, sodium chloride, potassium chloride and magnesium chloride. Commonly used organic solvents are ethanol, isopropanol and polyethylene glycol. Nucleic acid precipitation often contains a small amount of coprecipitated salt, which needs to be washed and removed with 70% to 75% ethanol. For low-density and bulky nucleic acid samples, they can be concentrated using solid polyethylene glycol or butanol prior to precipitation of the organic solvent.
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