Nucleic acid extraction is the first thing in molecular experiments and is the basis of almost all experiments. PCR, qPCR, library construction, and sequencing all require nucleic acids to proceed smoothly. The quality of nucleic acid extraction and purification is the prerequisite for ensuring the accuracy of subsequent detection data.
Nucleic acid is a type of biological macromolecule polymerized by nucleotides. It is one of the basic substances necessary for life. It mainly plays the role of a carrier of genetic information in living systems. It is mainly divided into DNA and RNA, among which RNA can be divided into rRNA, mRNA and tRNA according to different functions.
Nucleic acid extraction experiments involve two basic principles: cell lysis and nucleic acid precipitation.
1. Cell lysis is the process of releasing DNA from the cell wall and cell membrane. During this process, various physical and chemical means, such as freezing-crushing, enzymatic hydrolysis and ion exchange, can rupture cells and release nucleic acids;
2. Nucleic acid precipitation is the process of separating DNA from other impurities. The key trick here is to utilize a centrifuge and alcohol wash. The high-speed rotation of the centrifuge causes cell debris to settle at the bottom of the tube, while the alcohol helps the DNA form white floc on the tube wall, making it easier for us to extract.
Boiling cracking method
This method is generally used for manual extraction of DNA. Chromosomal DNA is much larger than plasmid DNA molecules, and chromosomal DNA is a linear molecule, while plasmid DNA is a covalently closed ring molecule. When the DNA solution is heated, linear chromosomal DNA is prone to denaturation, and covalently closed plasmid DNA is It restores its natural conformation when cooled; denatured chromosomal DNA fragments combine with denatured proteins and cell debris to form a precipitate, while the renatured supercoiled plasmid DNA molecules exist in a dissolved state in the liquid phase, so that the two can be separated by centrifugation.
Magnetic bead method
After improving and modifying the surface of superparamagnetic nanoparticles, superparamagnetic silicon oxide nanoscale magnetic beads are prepared. The magnetic beads can specifically bind to nucleic acid molecules. Utilizing the super paramagnetism of silica nanospheres, under the action of Chaotropic salts (guanidine hydrochloride, guanidine isothiocyanate, etc.) and an external magnetic field, DNA and RNA are isolated from blood, animal tissue, food, pathogenic microorganisms and other samples.
Phenol-chloroform extraction method
It mainly uses two different organic solvents to extract proteins to remove them alternately. When the homogenate is treated with phenol, since the bond between the protein and DNA has been broken, the surface of the protein molecules contains many polar groups that are similar to phenol and are soluble. At the same time, phenol inhibits the degradation of DNase, and the protein molecules dissolve in the phenol phase. DNA dissolves in the aqueous phase. After centrifugation:
Take out the aqueous layer and repeat the operation multiple times.
Combine the aqueous phases containing DNA.
Taking advantage of the insoluble nature of nucleic acids in alcohol, use ethanol to precipitate DNA.
After centrifugation, the DNA can be removed.
Anionic detergent method
DNA can be extracted directly from biological material by denaturing proteins with detergents such as SDS or sodium xylenate. Since DNA and proteins in cells are often bound by electrostatic attraction or coordination bonds, and anionic detergents can destroy this valence bond, anionic detergents are commonly used to extract DNA.
Spin column method
Special silicon matrix adsorption materials can specifically adsorb DNA, while RNA and proteins pass through smoothly. Then, high salt and low pH are used to bind nucleic acids, and low salt and high pH are eluted to separate and purify nucleic acids. Spin column purification is also a widely used method in kit extraction.
Thermal cracking alkali method
The alkaline lysis method is based on the difference in denaturation and renaturation of DNA to achieve the purpose of separation. Alkalinity denatures plasmid DNA, and then the pH value is adjusted to neutral to renature it. What is renatured is plasmid DNA, while chromosomal DNA does not renature and becomes tangled into a network material, which is removed by centrifugation.
Principle of CTAB method (classic method of plant DNA extraction)
CTAB (hexadecyltrimethylammoniumbromide) is a cationic detergent that can precipitate nucleic acids and acidic polysaccharides from low-ionic-strength solutions. In solutions with high ionic strength (>0.7 mol/L NaCl), CTAB forms complexes with proteins and polysaccharides but cannot precipitate nucleic acids. Through organic solvent extraction, impurities such as proteins, polysaccharides, and phenols are removed, and then ethanol precipitation is added to separate nucleic acids.
Other methods
In addition to the above commonly used methods, there are many others, such as ultrasonic, enzymatic hydrolysis, and concentrated salt methods.
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