Magnetic beads

Magnetic beads purification of DNA is mainly the use of interest exchange adsorption materials to adsorb nucleic acids, thereby separating nucleic acids and proteins and other substances in their cells. This article mainly outlines the principles of nucleic acid isolation and purification, the steps of nucleic acid separation and purification, and the principle of magnetic beads purification of DNA.

Principles of nucleic acid isolation and purification

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 isolation and purification steps

Most methods for nucleic acid isolation 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.

1. Cell lysis: Nucleic acids must be released from cells or other biological substances. 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 obtained by heating, adding a surfactant (SDS, Triton X-100, etc.) or a strong ionic agent (barium isothiocyanate, guanidine hydrochloride, creatinate). 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 chelate the metal ions Mg2+ necessary to maintain nuclease activity. Ca2+, 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. The lysozyme can catalyze the β-(1,4) bond hydrolysis between the proteoglycan N-acetylglucosamine and the N-acetylmuramic acid residue of the bacterial cell wall. Protease K catalyzes the hydrolysis of various polypeptide bonds, which retains enzymatic activity in the presence of EDTA, urea (1 to 4 mol/L) and detergent (0.5% SDS or 1% Triton X-100) at 65 ° C. It 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 choice of which method or methods can be determined based on the cell type, the type of nucleic acid to be separated, and the purpose of subsequent experiments.

2. Enzyme treatment: In the process of nucleic acid extraction, 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.

3. Isolation and purification of nucleic acids: The highly charged phosphate backbone of nucleic acids makes them more hydrophilic than other biological macromolecules such as proteins, polysaccharides, fats, etc., depending on their physicochemical properties, selective precipitation, chromatography, density The nucleic acid can be isolated and purified by a method such as gradient centrifugation.

(1) Phenol extraction/precipitation method: A classical method for nucleic acid separation is phenol:chloroform extraction. After the cells were lysed, the aqueous phase containing the nucleic acid was centrifuged, and an equal volume of a mixture of phenol:chloroform:isoamyl alcohol (25:24:1 by volume) 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. Hydrophobic proteins are assigned to the organic phase and nucleic acids are 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, 82 hydroxyquinoline is added during the preparation of the phenolic saturated liquid 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 precipitation with ethanol after extraction with phenol and chloroform. After adding pH 5.0-5.5 to the aqueous phase containing nucleic acid and NaOAc or KOAc at a final concentration of 0.3 M, sodium ions neutralize the nucleic acid phosphate backbone. Negative charge promotes hydrophobic renaturation of nucleic acids in an acidic environment. Then, 2 to 2.5 volumes of ethanol are added, and after a certain period of incubation, the nucleic acid can be effectively precipitated. Other organic solvents (isopropanol, polyethylene glycol (etc.) and salts (10.0 mol/L ammonium acetate, 8.0 mol/L lithium chloride, magnesium chloride, and low concentrations of zinc chloride, etc.). Ions can inhibit some enzymes or affect the precipitation and dissolution of nucleic acids, and should be selected in practical use. After centrifugation, the nucleic acid precipitates are rinsed with 70% ethanol to remove excess salts to obtain purified nucleic acids.

(2) Chromatography: Chromatography is a separation analysis method established by using differences in certain physical and chemical properties of different substances. Chromatography including adsorption chromatography, affinity chromatography, ion exchange chromatography and the like. It is widely used in the purification of nucleic acids because it is carried out simultaneously with separation and purification, and is supplied by a commercial kit. 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.

(3) Adsorption method: Glass powder or glass beads have been confirmed to be an effective nucleic acid adsorbent. In a high salt solution, the nucleic acid can be adsorbed onto a glass substrate, and the chaotropic salt sodium iodide or sodium perchlorate promotes the binding of the DNA to the glass matrix. 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 80% ethanol. Finally, the DNA bound to the glass beads is eluted with RNase-containing TE buffer, and the obtained DNA can be directly used for sequencing.

Magnetic beads purification of DNA principle

The magnetic bead nucleic acid purification technology uses nano-sized magnetic bead beads, and the surface of the magnetic bead beads is labeled with a functional group, which can react with the nucleic acid.

Magnetic Silica Particle refers to the surface of a magnetic bead coated with a layer of silicon material to adsorb nucleic acids. The purification principle is based on the purification method of glass milk.

Centrifugal magnetic beads refer to a material on the surface of the magnetic beads that is surrounded by a centrifugal exchange (such as DEAE, COOH) to achieve the purpose of adsorbing nucleic acids. The purification principles corresponding to magnetic bead beads of different properties are inconsistent. The biggest advantage of using magnetic beads to purify nucleic acids is automation. The magnetic beads can be aggregated or dispersed under magnetic magnetic conditions, so that the manual operation process such as centrifugation can be completely eliminated.

The plasmid DNA can also be isolated and purified 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/NaCl to adsorb the target DNA to the magnetic beads, and finally the magnetic field separates the adsorbed DNA and is washed with ethanol. Elution with TE for high yields of template DNA for capillary sequencing