Abstract: The separation and purification of nucleic acids is a basic technology in biochemistry and molecular biology. As molecular biology techniques are widely used in biology, medicine, and related fields, nucleic acid separation and purification technologies have been further developed. The emergence of various new methods and improved traditional classical methods and commercial reagent methods has greatly promoted the development of molecular biology. Traditional nucleic acid separation requires ethanol precipitation, phenol/chloroform extraction, centrifugation, and column chromatography. The operation is cumbersome, time-consuming and labor-intensive, and it is difficult to achieve automatic separation and purification. Glass powder or glass beads have proven to be an effective nucleic acid adsorbent. In high salt solutions, nucleic acids can be adsorbed onto a glass substrate to facilitate separation of nucleic acids. In addition, surface functionalized polymeric magnetic microspheres can be combined with biologically active substances and have broad application prospects in biology and medicine. The application of magnetic microspheres in nucleic acid separation accelerates the separation and automation of nucleic acids. Many methods have reported the preparation of magnetic microspheres. How to improve the specific binding of magnetic microspheres to DNA and reduce the adsorption of contaminants on the surface of microspheres plays an important role in promoting nucleic acid extraction technology of magnetic microspheres. In this paper, the principle and method of nucleic acid separation and purification will be discussed, and the automatic extraction technology of nucleic acid based on magnetic microspheres will be discussed in the preparation of magnetic microspheres, application in nucleic acid separation, separation and purification system based on magnetic microspheres.
The current status of development is reviewed and prospected.
Keywords: nucleic acid, magnetic microspheres, separation and purification, method
Principles and Steps for Nucleic Acid Separation
Nucleic acids are always bound to various proteins in cells. The separation of nucleic acids mainly refers to the separation of nucleic acids from biological macromolecules such as proteins, polysaccharides, and fats. The following principles should be followed when isolating nucleic acids: ensuring the integrity of the primary structure of the nucleic acid molecule; and eliminating other molecular contamination. Nucleic Acid Separation and Purification Steps Most methods for nucleic acid separation and purification generally include several major steps such as cell lysis, enzymatic treatment,separation of nucleic acids from other biological macromolecules, and nucleic acid purification. Each step can be implemented individually or in combination by a number of different methods.
u Cell Lysis
Nucleic acids must be released from cells or other biological materials. Cell lysis can be achieved by mechanical action, chemical action, enzymatic action, and the like.
(1) Mechanical action: including physical cracking methods such as hypotonic cracking, ultrasonic cracking, microwave cracking, freeze-thaw cracking and particle crushing. These methods use mechanical force to break cells, but mechanical forces can also cause breakage of nucleic acid strands, and thus are not suitable for the separation of high molecular weight long-chain nucleic acids.
(2) Chemical action: Under certain pH environment and denaturing conditions, the cells rupture, the protein denatures and precipitates, and the nucleic acid is released into the water phase. The above denaturing conditions can be achieved by heating, adding a surfactant (SDS, Triton X-100, Tween 20, NP-40, CTAB, sar-cosyl, Chelex-lO0, etc.) or a strong ionic agent (guanidine isothiocyanate, guanidine hydrochloride, muscle), obtained by sour. The pH environment is provided by the addition of a strong base (NaOH) or buffer (TE, STE, etc.). In a certain pH environment, surfactants or strong ionic agents can lyse cells, precipitate proteins and polysaccharides, and some metal ion chelators (EDTA, etc.) in the buffer can match the metal ions necessary for nuclease activity. Thereby inhibiting the activity of the nuclease and protecting the nucleic acid from degradation.
(3) Enzyme action: mainly by adding lysozyme or protease (proteinase K, plant protease or chain enzyme protease) to rupture cells and release nucleic acids. Proteases also degrade proteins that bind to nucleic acids and promote the separation of nucleic acids. Lysozyme can catalyze the hydrolysis of proteoglycans and cell walls of bacterial cell walls. Protease K catalyzes the hydrolysis of a variety of polypeptide bonds. Protease can retain high enzyme activity at high temperature and in the presence of appropriate amounts of EDTA, urea and detergent, which is beneficial to improve the extraction efficiency of high molecular weight nucleic acids. In practical work, enzyme action, mechanical action, and chemical action are often used in combination. The specific method can be determined according to the cell type, the type of nucleic acid to be separated, and the purpose of subsequent experiments.
u Enzyme Treatment
In the nucleic acid extraction process, the undesired substance can be degraded by adding an appropriate enzyme to facilitate the separation and purification of the nucleic acid. Adding proteases (proteinase K or chain enzyme protease) to the lysate can degrade proteins, inactivate nucleases (DNase and RNase), and DNase and RNase are also used to remove unwanted nucleic acids.
u Separation and Purification of Nucleic Acids
The highly charged phosphate backbone of nucleic acids makes them more hydrophilic than other biomacromolecules such as proteins, polysaccharides, fats, etc. According to their differences in physicochemical properties, nucleic acids can be separated by selective precipitation, chromatography, density gradient centrifugation, etc.
Several Important Nucleic Acid Separation Methods
u Phenol Extraction Method (Precipitation Method)
A classic method of nucleic acid separation is the phenol — chloroform extraction method. After the cells were lysed, the aqueous phase containing the nucleic acid was centrifuged, and an equal volume of phenol — chloroform: isoamyl alcohol mixture was added. Depending on the application, the two phases are mixed by vortexing (suitable for separating small molecular weight nucleic acids) or simply inverted (for separation of high molecular weight nucleic acids) and then centrifuged. The hydrophobic protein is assigned to the organic phase and the nucleic acid is retained in the upper aqueous phase. Phenol is an organic solvent that is previously saturated with STE buffer because the unsaturated phenol absorbs the aqueous phase and carries away a portion of the nucleic acid. Phenols are also susceptible to oxidative yellowing, while oxidized phenols can cause cleavage of phosphodiester bonds in the nucleic acid strand or cross-linking of nucleic acid strands; therefore, 8-hydroxyquinoline is added to the phenol-saturated solution to prevent oxidation of the phenol. Chloroform removes fat and denatures more protein, increasing extraction efficiency. Isoamyl alcohol reduces air bubbles generated during operation. The nucleoside salt can be precipitated by some organic solvents, and the nucleic acid can be concentrated by precipitation, changing the type of nucleic acid lysis buffer and removing certain impurity molecules. A typical example is ethanol precipitation after phenol and chloroform extraction. After adding NaOAc or KOAc to the nucleic acid-containing aqueous phase, sodium ions neutralize the negative charge on the nucleic acid phosphate backbone and promote the hydrophobic recovery of nucleic acids in an acidic environment. Then, ethanol is added, and after a certain period of incubation, the nucleic acid can effectively precipitate different ions to inhibit some enzymes or affect the precipitation and dissolution of nucleic acids, and should be selected in actual use. After centrifugation, the nucleic acid precipitate was rinsed with 70% ethanol to remove excess salt to obtain a purified nucleic acid.
u Density Gradient Centrifugation
Density gradient centrifugation is also used for the separation and analysis of nucleic acids. Double-stranded DNA, single-stranded DNA, RNA, and proteins have different densities, so they can form pure sample bands of different densities by density gradient centrifugation. This method is suitable for the preparation of a large number of nucleic acid samples, in which cesium chloride-bromination Ethidium gradient equilibrium centrifugation is considered the preferred method for purifying large amounts of plasmid DNA. Barium chloride is a standard medium for density gradient centrifugation of nucleic acid. The ethidium bromide in the gradient solution is combined with the nucleic acid. The nucleic acid band formed by centrifugation is irradiated by ultraviolet light to generate fluorescence, which is detected and puncture is recovered by injection needle. Purified nucleic acid is obtained by dialysis or ethanol precipitation to remove cesium chloride.
u Chromatography
Chromatography is a separation analysis method established by using differences in certain physical and chemical properties of different substances, including adsorption chromatography, affinity chromatography, ion exchange chromatography and the like. This method is widely used for the purification of nucleic acids due to the simultaneous separation and purification, and the availability of commercial kits.
l Ion exchange chromatography
The method uses a substance having ion exchange properties as a stationary phase, which is reversibly exchanged with ions in the mobile phase, thereby being capable of separating the ionic compound. The nucleic acid is purified by ion exchange chromatography because the nucleic acid is a highly negatively charged linear polyanion. In a low ionic strength buffer, the negatively charged nucleic acid is bound by the electrostatic reaction of the target nucleic acid with the functional matrix on the anion exchange column. The impurity molecules are eluted onto the positively charged substrate. The ionic strength of the buffer is then increased, the nucleic acid is eluted from the substrate, and the purified nucleic acid is obtained by precipitation with isopropanol or ethanol. This method is suitable for the purification of large-scale nucleic acids.
l Affinity chromatography
This method utilizes the specific affinity of the substances to be separated and their specific ligands to achieve separation.
Chandler, a scholar, reported a method for isolating nucleic acids using peptide nucleic acids (PNA). PNA is a class of DNA analogs based on N-(2-aminoethyl)-glycine structural units and can be used as a reagent for purifying picogram-grade ribosomal DNA (rDNA) and ribosomal RNA (rRNA). In this method, biotin-labeled peptide nucleic acid is used as a probe, and magnetic beads coated with streptavidin are used as a solid phase carrier. The PNA probe is mixed with the nucleic acid of interest (DNA or RNA) in a high salt environment. After the boiling, ice bath, and incubation hybridization steps, the paramagnetic particles coated with streptavidin are directly added, and the PNA-nucleic acid hybrid is captured by standing and washed with water to obtain a purified nucleic acid.
Another experimental team also used the principle of affinity chromatography to successfully separate plasmid DNA using a triple helix DNA. The triploid DNA consists of a homogenous purine-homopyrimidine double helix chain and a homopyrimidine single strand. The T on the single strand recognizes the A·T base pair to form a T·A·T triplet, a protonated single-stranded cell. Pyrimidine C recognizes the G·C base pair to form a C·G·C triplet. Under appropriate conditions, the combination of triploid has high specificity and high stability. The ligand polypyrimidine oligonucleotide strand is chemically linked to Sephacryl S-1000SF particles to form an affinity vector. When the plasmid DNA solution containing the desired sequence is mixed with it, the plasmid is bound to the affinity carrier particle in an acidic environment (pH 4.5–5.5), and the high concentration of NaCl in the solution stabilizes the triplet form and reduces the protein and cells non-specific binding of DNA. After a period of reaction, the particle suspension is added to the column, and the pH is adjusted to an alkaline environment with an appropriate eluent to depolymerize the triplet and the plasmid is eluted. By separating the plasmid DNA by this method, the plasmid yield can reach 62% of the added amount.
l Adsorption by magnetic beads
Under certain ionic conditions, nucleic acids can be selectively adsorbed onto silica, silica gel or glass surfaces to be separated from other biomolecules. Other selective adsorption methods use modified or coated magnetic beads as solid support, magnetic beads can be separated by magnetic field without centrifugation, and nucleic acids bound to the solid support can be eluted with low salt buffer or water. The method separates and purifies nucleic acid, and has the advantages of good quality, high yield, low cost, quickness, simplicity, labor saving, and easy automation.
Glass powder or glass beads have proven to be an effective nucleic acid adsorbent. In high salt solutions, nucleic acids can be adsorbed onto a glass substrate, and sodium iodide or sodium perchlorate promotes binding of the DNA to the glass matrix. Dederich et al. isolated and purified nucleic acids with pickled glass beads to obtain high yields of plasmid DNA. In this method, the cells are lysed in an alkaline environment, and the lysate is neutralized with potassium acetate buffer, and directly added to a glass bead filter plate containing isopropyl alcohol, and the plasmid DNA precipitated by isopropyl alcohol is bound to the glass beads. Cell debris and protein pellets were removed by vacuum extraction with 8O ethanol. Finally, the DNA bound to the glass beads is eluted with RNaseA-containing TE buffer, and the obtained DNA can be directly used for sequencing.
With the development of various synthesis and modification methods, magnetic microsphere-based separation techniques have been widely used in biological separation, such as separation of cells, DNA, RNA, proteins, and the like. In 1997, Hawkins et al. proposed a DNA extraction technology based on magnetic particles. The application of magnetic microspheres in nucleic acid separation has gradually formed a new research hotspot. Magnetic microspheres extract DNA not only has the characteristics of simple, rapid and effective, but also avoids centrifugation and extensive use of organic solvents. It is a new nucleic acid extraction technology with economical and potential application. At the same time, the use of magnetic composite microspheres in nucleic acids greatly improves the efficiency of separation and purification of nucleic acids, and provides technical support for the automated extraction of nucleic acids.
Percin et al. studied the extraction of plasmid DNA from polyhydroxyethyl methacrylate magnetic composite nanospheres in E. coli lysate. The experimental results show that the magnetic nanoparticles have high selectivity to plasmid DNA. Polyimine coated super paramagnetic Fe3O4 magnetic microspheres for rapid extraction of bacterial plasmid DNA. The results show that the nucleic acid extracted by the polymer magnetic microsphere has high purity. The surface carboxylated magnetic microspheres can be used for separation and purification of mRNA and supercoiled plasmid DNA. The magnetic natural polymer microspheres are obtained by coating magnetic particles with gelatin and used for the separation of bacterial genomic DNA.
Elkin et al. isolated and purified plasmid DNA using carboxylated magnetic beads. After the cell is lysed, the aqueous phase containing the plasmid is centrifuged, and then the carboxylated magnetic particles are added, and then precipitated with PEG/NaC1 to adsorb the target DNA to the magnetic beads. Finally, the adsorbed DNA is separated by a magnetic field, and the ethanol is separated. Washing, eluting with water, yields high yields of template DNA suitable for capillary sequencing. It is also useful to use iron particles as a solid support to purify plasmid DNA by magnetic field separation. The bacteria are lysed by lysozyme-boiling, and the plasmid is released into the suspension, captured by iron beads, and the iron beads are separated by a magnetic field. After washing, the plasmid is eluted with water to obtain high-yield, sequencing-grade plasmid DNA.
Prospects for Nucleic Acid Separation
With the advent of the “post-genome era”, rapid and efficient techniques such as capillary electrophoresis have been widely used in nucleic acid analysis. In the past, some traditional nucleic acid extraction methods have not adapted to the development requirements of molecular biotechnology due to cumbersome operation, low extraction efficiency, time-consuming and laborious use, or the use of toxic chemical reagents, and the difficulty of realizing automated instrument operation. With the efforts of people and the advancement of technology, it is believed that there will be more simple, safe, efficient, low-cost and new methods for nucleic acid separation and purification for automated instrumentation, which will promote the development of molecular biology more quickly.
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