Summary of nucleic acid (RNA and DNA) extraction methods

Nucleic acid is a biological macromolecular compound formed by the polymerization of many nucleotides. It is one of the most basic substances of life. Nucleic acid exists in all animal cells, plant cells, microorganisms, and organisms. Nucleic acids often combine with proteins to form nuclear proteins. Different nucleic acids have different chemical composition and nucleotide sequence. Nucleic acid mainly includes RNA (ribonucleic acid) and DNA (deoxyribonucleic acid), among which DNA is the main material basis for storing, copying and transmitting genetic information.

The principle of nucleic acid extraction is to first ensure the integrity of the primary structure of the accounting, and secondly, to exclude the contamination of other molecules.

When extracting nucleic acid, attention should be paid to simplify the steps and shorten the extraction time; reduce the degradation of nucleic acid by chemical factors; reduce the degradation of nucleic acid by physical factors: mechanical shearing force and high temperature; prevent the biological degradation of nucleic acid.

Nucleic acid extraction is roughly divided into three parts: crushing, extraction, and purification

Cell lysis

  1. Physical method: boiling method, glass bead method, ultrasonic method, grinding method, freeze-thaw method, homogenization method
  2. Chemical method: Surfactant (SDS method), alkaline lysis method
  3. Biological method: enzymatic method (lysozyme, proteinase K, etc.)

Summary of common sample extraction methods

Concentrated salt method: use the different solubility of RNA and DNA in salt solution to separate the two

Organic solvent extraction method: organic solvent acts as a protein denaturant while inhibiting the degradation of nucleases

High gradient centrifugation: use the principle of different content density to separate various content

Adsorption material binding method: ①Silicone material binds nucleic acid with high salt and low pH value, and eluates with low salt and high pH value. ②Anion exchange resin binds nucleic acid with low salt and high pH value, and eluates with high salt and low pH value. ③Magnetic bead particle coating Different genes can adsorb different targets to achieve the purpose of separation

Guanidine extraction combined with silica adsorption method

Silica has the characteristic of specifically adsorbing nucleic acids. Guanidine isothiocyanate can lyse cells. Therefore, in the presence of high concentrations of guanidine isothiocyanate, nucleic acid components released from cells including bacteria or virus particles can bind to the dioxide On silicon, this feature can be used to extract DNA and RNA from blood and urine

TRlzol reagent to extract RNA

Lyse the cells with TRlzol reagent and add chloroform. After centrifugation, three layers can be seen. The upper layer is a transparent water phase containing RNA, the middle contains DNA, and the lower layer is a red organic phase containing protein.

Spin column extraction

Spin column technology is a relatively simple method for the separation and purification of trace nucleic acids. It is a kind of silicon adsorption method and can be divided into three parts in principle:

①Using the lysis solution to promote cell disruption and release the nucleic acid in the cell.

② The released nucleic acid is specifically adsorbed on a specific silicon carrier. This carrier only has a strong affinity and adsorption power for nucleic acid, and basically does not adsorb other biochemical components such as proteins, polysaccharides, and lipids, so it is Throw out the pillar.

③ The nucleic acid adsorbed on the specific carrier is eluted with the eluent, and the purified nucleic acid is separated and purified.

Magnetic Bead Method

According to the same principle as the silica gel membrane spin column, the surface of superparamagnetic nanoparticles is modified and modified by nanotechnology to prepare superparamagnetic silica nanomagnetic beads. The magnetic beads can specifically recognize and efficiently bind with nucleic acid molecules on the microscopic interface. Utilizing the superparamagnetism of silica nanospheres, under the action of Chaotropic salts (guanidine hydrochloride, guanidine isothiocyanate, etc.) and an external magnetic field, DNA and RNA in blood, animal tissue, food, pathogenic microorganisms and other samples can be collected After separation, it can be used in various fields such as clinical disease diagnosis, blood transfusion safety, forensic identification, environmental microbial testing, food safety testing, molecular biology research, etc.