What are the nucleic acid purification technologies?
At present, nucleic acid purification technologies widely used in scientific research can be divided into two categories: those that use media and those that do not use media. If media is used, nucleic acid is separated from all other impurities at one time; if media is not used, the first step is to separate the nucleic acid from all other impurities. Nucleic acid and salt are separated from macromolecular impurities, and nucleic acid is separated from salt by precipitation of nucleic acid.
1) Classical purification technology using phenol/chloroform extraction
After the cells are lysed, the aqueous phase containing nucleic acid is separated by centrifugation, and an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1 volume) mixture is added. According to the application purpose, the two phases are vortexed and mixed (suitable for the separation of small molecular weight nucleic acids) or simply inverted and mixed (suitable for the separation of high molecular weight nucleic acids) and then centrifuged. Hydrophobic proteins are partitioned into the organic phase, and nucleic acids are retained in the upper aqueous phase.
Phenol is an organic solvent. It should be saturated with STE buffer beforehand. Unsaturated phenol will absorb the water phase and take away a part of nucleic acid. Phenol is also easy to oxidize and turn yellow, and oxidized phenol can cause the phosphodiester bond in the nucleic acid chain to break or crosslink the nucleic acid chain; therefore, a special substance should be added when preparing the phenol saturated solution to prevent phenol oxidation. Chloroform can remove fat and denature more protein, thereby improving extraction efficiency. Isoamyl alcohol can reduce bubbles generated during operation.
2) Purification technology using ion exchange media
The lysate is passed through the column, and the nucleic acid is bound to the ion exchange medium; after washing to remove residual impurities, the nucleic acid is eluted from the medium with a high-salt buffer. After standard ethanol/isopropanol precipitation, ethanol washing, drying and other operations, the pure nucleic acid is obtained and dissolved in a suitable buffer.
3) Purification technology using adsorption media
The lysate is passed through the column, and the nucleic acid is selectively adsorbed by the adsorption medium; after washing to remove residual impurities, the nucleic acid is eluted from the medium with water or a suitable low-salt buffer, and it can be directly used in subsequent experiments.
4) Density gradient centrifugation
Density gradient centrifugation is also used for the separation and analysis of nucleic acids. Double-stranded DNA, single-stranded DNA, RNA and protein have different densities, so pure sample zones of different densities can be formed by density gradient centrifugation. This method is suitable for the preparation of a large number of nucleic acid samples, in which cesium chloride 2 ethyl bromide Spindle gradient equilibrium centrifugation is considered to be the preferred method for purification of large amounts of plasmid DNA. Cesium chloride is the standard medium for nucleic acid density gradient centrifugation. The ethidium bromide in the gradient solution combines with nucleic acid. The nucleic acid zone formed after centrifugation is irradiated by an ultraviolet lamp to produce fluorescence and be detected. Dialysis or ethanol precipitation removes cesium chloride to obtain purified nucleic acid.
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