Nucleic acid: physical and chemical properties, purification principle requirements, extraction method introduction
Nucleic acid is the basis of molecular biology, and nucleic acid extraction is the threshold that the entire molecular industry cannot bypass. In many cases, the quality of nucleic acid extraction from a sample directly determines the validity of the test results. 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 polybasic acid. A neutral or alkaline buffer can be used to dissociate nucleic acid into anions and move towards the anode in an electric field. This is the principle of electrophoresis.
The chemical properties of nucleic acids
①Acid effect: Under strong acid and high temperature, nucleic acid is completely hydrolyzed into bases, ribose or deoxyribose and phosphoric acid. In the slightly dilute inorganic acid, the most easily hydrolyzed chemical bond is selectively broken, usually the glycosidic bond connecting purine and ribose, thereby producing apurinic nucleic acid.
②Alkaline effect
1. DNA: When the pH value exceeds the physiological range (pH7~8), it will have a more subtle effect on the DNA structure. The base effect changes the tautomeric state of bases. This change affects the hydrogen bonding between specific bases, resulting in the dissociation of DNA double strands, which is called DNA denaturation
2. RNA: At higher pH, the same denaturation occurs in the helical region of RNA, but it is usually masked by alkaline hydrolysis of RNA. This is because the 2'-OH in RNA participates in the intramolecular attack on the phosphate molecules in the phospholipid bond, leading to the breakage of RNA.
③Chemical denaturation: Some chemical substances can denature DNA/RNA under neutral pH. The energy stability of the nucleic acid secondary structure formed by the stacked hydrophobic clips is weakened, and the nucleic acid is denatured.
Physical properties of nucleic acids
①Viscosity: DNA's high axial ratio and other properties make its aqueous solution highly viscous. Long DNA molecules are easily damaged by mechanical force or ultrasound, and the viscosity decreases.
② Buoyancy density: DNA can be purified and analyzed according to its density. In a high concentration molecular weight salt solution (CsCl), DNA has approximately the same density as the solution. Centrifuge the solution at a high speed, and the CsCl tends to settle to the bottom, thereby establishing a density gradient, and the DNA finally settles to its buoyancy density. Position, forming narrow bands, this technique is called equilibrium density gradient centrifugation or isocratic gradient centrifugation.
③Stability: The structure of nucleic acid is quite stable. The main reasons are 1. The hydrogen bond between base pairs 2. The accumulation of bases 3. The cations in the environment.
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 protein, polysaccharides and lipids as much as possible
Points for attention in nucleic acid extraction
1. Simplify the steps and shorten the extraction time
2. Reduce the degradation of nucleic acids by chemical factors
3. Reduce the degradation of nucleic acids by physical factors: mechanical shearing force and high temperature
4. Prevent the biodegradation of nucleic acids
Extraction type
1. Total RNA extraction
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 nuclear 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 genetic diagnosis, it is usually required that the length of the fragment obtained 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 the 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.
The main steps of nucleic acid extraction
1. Lyse cells
Remove proteins that are bound to nucleic acids, polysaccharides, lipids and other biological macromolecules, remove other unwanted nucleic acid molecules, such as when extracting DNA molecules, remove RNA, and vice versa.
2. Precipitating nucleic acid
Purify nucleic acids, remove impurities such as salts and organic agents
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 in the organic phase, and proteins are located between the two phases.
The classic method of DNA extraction is the so-called phenol-chloroform extraction method. Because it is easier to remove the protein by using two different organic solvents alternately, the extraction order is phenol, phenol/chloroform (1:1), and chloroform. The DNA extracted by this method has high purity, large fragments and good effect. The disadvantages are More cumbersome.
Note: When recovering the upper water phase, be sure not to touch the interface of the two phases to avoid sucking proteins and other substances into the new centrifuge tube. For DNA precipitation, absolute ethanol is the organic solvent of choice. Another: isopropanol, sodium acetate, etc. The purpose of rinsing DNA with 70% ethanol is to remove residual salts and excess SDS and phenols. Because SDS remains dissolved in 70% ethanol and does not co-precipitate with DNA, this can be removed by discarding the supernatant. Detergent to avoid the impact on future PCR reactions. TE buffer: 10mmol/L Tris-Hcl 1mmol/L EDTA pH 8.5 or 8
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. Chromatographic column method
Spinning 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. Although spin columns on the market have their own characteristics, they can usually be divided into three parts in principle:
(1) Use the lysate to break the cell 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. It was thrown out of the column during centrifugation.
(3) The nucleic acid adsorbed on the specific carrier is eluted with the eluent, and the purified nucleic acid is separated.
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
The DNA solution is heated 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 silica nanomagnetic beads. 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. 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 in blood, animal tissue, food, pathogenic microorganisms and other samples can be collected Isolated, can be used in clinical disease diagnosis, blood transfusion safety, forensic identification, environmental microbial testing, food safety testing, molecular biology research and other fields.
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.