Introduction of common nucleic acid extraction and purification methods

Introduction of common nucleic acid extraction and purification methods

1. Phenol chloroform extraction method

Phenol chloroform is used to extract DNA by using phenol as a denaturant of protein, repeated extraction to denature the protein, SDS (sodium dodecyl sulfonate) to lyse the cell membrane, and digest proteins or polypeptides or small peptides in the presence of proteinase K and EDTA. Molecules that degrade nucleoproteins by denaturation, freeing DNA from nucleoproteins. DNA is soluble in water but insoluble in organic solvents.

The surface of the protein molecule has a hydrophilic group, which is also easy to perform hydration, and a hydration layer is formed on the surface so that the protein molecule can smoothly enter the aqueous solution to form a stable colloidal solution. In the presence of organic solutions, this colloidal stability of proteins is disrupted, denaturing and precipitation. After centrifugation, the organic solvent is in the bottom (organic phase) of the test tube, the DNA is present in the upper aqueous phase, and the protein is precipitated between the two phases. Using the extraction principle, according to the protein-nucleic acid is dissolved in different reagent layers, the pipette tip is inserted into different liquid layers to extract the required components, and purified nucleic acid is obtained after multiple items of washing.

The disadvantage of the phenol-chloroform extraction method is that due to the use of phenol, chloroform and other reagents, the toxicity is high, and long-term operation has a great impact on personnel health, and the recovery rate of nucleic acid is low and the loss is large. Due to the large operating system, Different experimenters have poor repeatability, which is not conducive to the protection of RNA, and it is difficult to perform micro-quantification operations.

The advantage of the phenol-chloroform extraction method is that it uses common reagents and medicines in the laboratory, and the cost is relatively low.

2. Spin column purification

The method of spin column purification is to use the surface of the nucleic acid to be covered with a film composed of water molecules. In a high-salt environment, this layer of hydrophilic film will be destroyed so that the nucleic acid can be adsorbed on the spin column, and other impurities such as proteins. , metabolites, etc. will be separated by centrifugal precipitation and nucleic acid. Finally, by elution, the nucleic acid is detached from the adsorption membrane, and the purified nucleic acid can be obtained.

Spin column purification is also a widely used method for kit extraction.

But even so, the spin column purification method still has disadvantages:

Highly dependent on the binding capacity of the adsorption membrane;

Incomplete elution leads to loss of nucleic acid;

Nucleic acid cannot be extracted in large quantities.

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