As the foundation of all molecular biology research, nucleic acid extraction is always the first step in a research. The quality of the extracted nucleic acid is the key to the success or failure of downstream molecular biology experiments. This article will introduce in detail the relevant information about DNA extraction, including the principles of DNA extraction, precautions for the extraction process, recommended methods for extracting DNA from different types of samples, and so on.
Basic principles of DNA extraction
DNA extraction can be simply divided into two major steps: lysis and purification. Lysis is the process of destroying the cell structure of the sample so that the DNA in the sample is freed in the lysis system. Purification is the process of making the DNA and other components in the lysis system, such as The process of complete separation of protein, salt and other impurities.
Cracking process
Conventional lysis solutions contain detergents (such as SDS, Triton X-100, NP-40, Tween 20, etc.) and salts (such as Tris, EDTA, NaCl, etc.).
The role of detergent
Denature protein
Destroy the membrane structure;
Remove proteins that interact with nucleic acids.
The role of salt
Provide a suitable lysis environment, such as Tris;
Inhibit the degradation of nucleic acid by nuclease, such as EDTA;
Maintain the stability of nucleic acid structure, such as NaCl.
Protease may also be added to the lysis system to digest the protein into small fragments to promote the separation of DNA and protein. At the same time, it is also convenient for subsequent purification operations.
Purification process
Phenol chloroform extraction
The method consists of two steps:
Use phenol chloroform to repeatedly extract the lysis system to remove protein and realize the separation of DNA and protein;
The DNA is then precipitated with alcohol to achieve the separation of nucleic acid and salt.
Phenol-chloroform extraction is an effective method to remove protein, but if the protein content exceeds its saturation, the protein in the lysis system will not be removed at one time, and multiple repeated extractions are required, and each extraction will Lead to the loss of nucleic acid.
The biggest advantage of phenol-chloroform extraction is low cost and low requirements for experimental conditions; this is one of the methods, and automatic nucleic acid extraction systems-DNA extractor and RNA extractor have also been developed on the market.
High salt precipitation
The high-salt precipitation method is a variant of the phenol-chloroform extraction method, which omits the trouble of the phenol-chloroform extraction operation, and almost overcomes all the shortcomings of the phenol-chloroform extraction method, but the purity of the obtained DNA is not stable enough.
Spin column purification
This method uses certain solid-phase media to selectively adsorb nucleic acids but not proteins and salts under certain specific conditions to achieve the separation of nucleic acids from proteins and salts. It is currently a widely used method for kit extraction.
This method is less affected by man-made operating factors, and the purity and stability of the extracted DNA is very high. Its disadvantage is that when the sample is excessive, it needs to be centrifuged repeatedly, and the extraction efficiency of the sample is low.
Magnetic Bead Method
In this method, the purification medium is coated on the surface of nano-scale magnetic beads, and the DNA is attached to the magnetic beads and moved directionally under the action of an external magnetic field through the medium's adsorption of DNA, so as to achieve the purpose of separating nucleic acid from other substances.
Compared with other centralized methods, the magnetic bead method has unparalleled advantages, including:
High extraction sensitivity (only a small amount of samples are required);
The purity of the purification is high (the nucleic acid can be completely separated from the impurities);
High extraction yield (each mg magnetic beads can adsorb 500μg DNA);
Fast separation speed (magnetic separation only takes a few seconds);
Automated operation (automatically complete the operation process through the machine, without manpower);
High-throughput extraction (can complete the extraction of hundreds of samples at the same time);
Non-toxic, harmless and non-polluting (the reagent does not contain any toxic substances).
This method relies on a magnetic separation device or an automatic extractor, and the current price is still very high, and it is not suitable for routine experiments in small laboratories.
