Nucleic acid is the carrier of genetic information, the most important biological information molecule, and the main object of molecular biology research. Therefore, nucleic acid extraction is the most important and basic operation in molecular biology experimental technology.
Nucleic acid is divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). RNA can be divided into ribosomal RNA (rRNA), messenger RNA (mRNA) and transfer RNA (tRNA) according to their functions.
DNA is mainly concentrated in the nucleus, mitochondria and chloroplasts, while RNA is mainly distributed in the cytoplasm.
In nucleic acids, purine bases and pyrimidine bases have conjugated double bonds, so nucleic acids have ultraviolet absorption characteristics. The ultraviolet absorption of DNA sodium salt is around 260nm, and its absorbance is represented by A260. It is in the absorption trough at 230nm, so ultraviolet spectroscopy can be used. The photometer performs quantitative and qualitative determination of nucleic acids.
Nucleic acid is an amphoteric electrolyte, which is equivalent to a polyacid. A neutral or alkaline buffer can be used to dissociate nucleic acid into anions, which are placed in an electric field and move towards the anode. This is the principle of electrophoresis.
Principles and requirements of nucleic acid extraction and purification
1. Ensure the integrity of the primary structure of nucleic acid;
2. Eliminate pollution from other molecules (such as eliminating RNA interference when extracting DNA);
3. There should be no organic solvents and high concentrations of metal ions that can inhibit enzymes in nucleic acid samples;
4. Minimize macromolecular substances such as proteins, polysaccharides and lipids as much as possible.
Nucleic acid extraction type
1. Extraction of total RNA
Of the total RNA, 75-85% is rRNA (mainly 28S-26S/23S and 18S/16S rRNA), and the rest consists of mRNA and small RNA with different molecular weights and nucleotide sequences such as tRNA, 5S rRNA, 5.8S rRNA, miRNA, siRNA, small nuclear RNA (small nuclear RNA, snRNA) and small nucleolar RNA (small nuceolar RNA, snoRNA) and other components.
2. miRNA extraction
MicroRNAs (miRNAs) are small, highly conserved RNA molecules, such as small interfering RNAs (siRNAs), which regulate the expression of their homologous mRNA molecules by base pairing with them to prevent expression through various mechanisms. They have become a key regulatory agency for development, cell proliferation, differentiation and cell cycle.
3. Genomic DNA extraction
For gene structure and function research and gene diagnosis, it is usually required that the length of the obtained fragment is not less than 100-200kb. In the DNA extraction process, various factors that cause DNA fragmentation and degradation should be avoided as much as possible to ensure the integrity of DNA and lay the foundation for subsequent experiments.
4. Plasmid extraction
The plasmid extraction method is to remove RNA, separate the plasmid from the bacterial genomic DNA, and remove proteins and other impurities to obtain a relatively pure plasmid.
Nucleic acid extraction and purification method
1. Phenol/chloroform extraction method
Invented in 1956, after phenol/chloroform treatment of cell crushing liquid or tissue homogenate, nucleic acid components mainly composed of DNA are dissolved in the aqueous phase, and lipids are mainly contained in the organic phase, and proteins are located between the two phases.
2. Alcohol precipitation method
Ethanol can eliminate the hydration layer of nucleic acid and expose the negatively charged phosphate groups. Positively charged ions such as NA﹢ can combine with the phosphate groups to form a precipitate.
3. Chromatography column method
Through the special silicon matrix adsorption material, DNA can be specifically adsorbed, and RNA and protein can pass through smoothly, and then use high salt and low pH to bind nucleic acid, and low salt and high pH elution to separate and purify nucleic acid.
4. Thermal cracking alkali method
Alkaline extraction mainly uses the topological difference between covalently closed circular plasmids and linear chromatin to separate them. Under alkaline conditions, denatured proteins are soluble.
5. Boiling cracking method
Heat the DNA solution to use the characteristics of linear DNA molecules to separate the DNA fragments from the precipitate formed by denatured proteins and cell debris by centrifugation.
6. Nano magnetic bead method
The surface of superparamagnetic nanoparticles is modified and modified by nanotechnology to prepare superparamagnetic silicon oxide nanomagnetic beads. The magnetic beads can specifically recognize and efficiently combine with nucleic acid molecules on the microscopic interface. Using the superparamagnetism of silica nanospheres, under the action of Chaotropic salts (guanidine hydrochloride, guanidine isothiocyanate, etc.) and an external magnetic field, DNA and RNA are separated from blood, animal tissues, food, pathogenic microorganisms and other samples .
7. Other methods
In addition to the above-mentioned commonly used methods, there are multiple methods such as ultrasound, repeated freezing and thawing, enzymatic hydrolysis, and hypotonic lysis.