The principle and steps of the magnetic bead method of separating purified DNA.

related topics
magnetic bead purification
DNA
is mainly the use of interest exchange adsorption material adsorption nucleic acids, so as to isolate nucleic acids and
proteins
and other substances in their cells. This paper mainly summarizes the principle of purified DNA by magnetic bead method, the principle of nucleic acid separation and purification, and the steps of nucleic acid separation and purification.Magic bead method purified DNA principle magnetic bead method nucleic acid purification technology using nano-scale bead microbeads, the surface of this magnetic bead microbead marked a kind of official energy group, can be adsorption reaction with nucleic acid. Magnetic Silica Particle refers to the magnetic bead microbead surface wrapped in a layer of silicon material to absorb nucleic acids, the purification principle is typed in the way glass milk purification.
centrifugal beads are those that are wrapped in a layer of materials (such as DEEAE, COOH) that can be centrifugally exchanged on the surface of the bead microbeads, so as to achieve the purpose of adsorption of nucleic acids. The principle of purification corresponding to magnetic bead microbeads of different properties is inconsistent. The greatest advantage of using the bead method to purify nucleic acids is automation. Beads can aggregation or dispersion under magnetic field conditions, allowing for a complete escape from manual processes required for centrifugation. Omega has a comprehensive beaded nucleic acid separation
-reagent
box, based on this technology, with a 'Mag-Bind' before the name.principle of nucleic acid separation and purification nucleic acids are always combined with a variety of proteins in cells. The separation of nucleic acids mainly refers to the separation of nucleic acids from proteins, polysaccharides, fats and other biometric substances. The following principles should be followed when separating nucleic acids: to ensure the integrity of the nucleic acid molecular structure: to eliminate other molecular contamination.Nucleic acid separation and purification steps Most nucleic acid separation and purification methods generally include cell lysation, enzyme treatment, nucleic acid and other biological polymer material separation, nucleic acid purification and other major steps. Each step can be implemented individually or jointly by a number of different methods..1. Cell cleavage: Nucleic acids must be released from cells or other biological substances. Cell lysation can be achieved by mechanical, chemical, enzyme and other methods.(1) mechanical effects: including low-permeability, ultrasonic cracking, microwave cracking, freeze-thawing and particle fragmentation and other physical cracking methods. These methods use mechanical force to break cells, but mechanical force can also cause the rupture of nucleic acid chain, and therefore not suitable for the separation of high molecular weight long chain nucleic acids. It has been reported that the length of nucleic acid fragments extracted by ultrasonic lysate is between < 500bp and > 20kb, while nucleic acid extracted by particle homogeneity is generally < 10kb.(2) chemical effects: cell rupture, protein degeneration precipitation, nucleic acid release to the water phase under certain p H environment and denaturation conditions. The above denaturation conditions can be obtained by
heating
, adding
surfactants
(
SDS
, Triton X-100, Tween 20, NP-40, CTAB, sar-cosyl, Chelex-100, etc.) or strong ions (isothionate, hydrochloric acid, creatonin). The p H environment is provided by the added strong alkali (NaOH) or buffer (TE, STE, etc.). In a certain p H environment, surfactants or strong ion agents can make cell lysate, protein and polysaccharide precipitation, buffer some metal ion chelating agents (EDTA, etc.) can chelate the activity of nucleases necessary metal ions Mg2, Ca2 plus, thereby inhibiting the activity of nucleases, to protect nucleic acids from degradation.(3) enzyme action: mainly by adding lysolysase or protease (protease K, plant protease or chainase protease) to break the cell, nucleic acid release. Proteases also degrade proteins that bind to nucleic acids, facilitating the separation of nucleic acids. Among them, lysolysase can catalyz the protein polysaccharide N-acetyl glucosamine and N-acetyl cell wall acid residue between the β-(1,4) bond hydrolysis. Protease K catalyzes hydrolysis of a variety of
peptide
bonds, which retain enzyme activity at 65 degrees C and with EDTA, urea (1 to 4mol/L) and detergents (0.5% SDS or 1 %Triton X-100), which helps to improve the extraction efficiency of high molecular nucleic acids. In practice, enzyme, mechanical and chemical effects are often used in a joint effort. Which or which methods can be selected can be determined according to the cell type, the
acid type to be
isolated, and the purpose of subsequent experiments..2. Enzyme treatment: During nucleic acid extraction, unwanted substances can be degraded by adding appropriate enzymes to help the separation and purification of nucleic acids. The addition of proteases (protease K or strepase proteases) to lysates can degrade proteins, inactivate nucleases (DNase and RNase), and DNase and RNase are also used to remove unwanted nucleic acids..3. Separation and purification of nucleic acids: the high-charged phosphate skeleton of nucleic acids makes them more hydrolysophate than proteins, polysaccharides, fats and other biological polymer substances, according to their physical and chemical properties, using selective precipitation, layering, density gradient centrifugation and other methods can separate and purify nucleic acids.
(1) phenol extraction/precipitation method: a classic method of nucleic acid separation is phenol-chloroform extraction. After cell lysing, centrifuges the water phase containing nucleic acids and adds an equal volume of phenols: chloroform: isoquinol (25:24:1 volume) mixture. According to the application purpose, the two phases are mixed by vortex oscillation (suitable for separating small molecular weight nucleic acids) or simply reverse mixing (for separating high molecular weight nucleic acids) after centrifugal separation. Hydrophobic proteins are assigned to the organic phase, while nucleic acids are retained in the upper water phase. Phenol is an organic solvent that is saturated in advance with a STE buffer and takes away some of the nucleic acids as unsaturated phenols absorb the water phase. Phenols are also prone to oxidation yellowing, and oxidized phenols can cause the nucleic acid chain of phosphate phosphate bonds to break or make the nucleic acid chain cross-link: therefore, in the preparation of phenol saturation solution to add 82 hydroxyquinone to prevent phenol oxidation.
chloroform removes fat and denatures more proteins, improving extraction efficiency. Isosterol reduces the bubbles produced during operation. Nucleates can be precipitated by some organic solvents to concentrate nucleic acids, change the type of nucleic acid dissolved buffer and remove certain impurity molecules. A typical example is the precipitation of ethanol after phenol and chloroform pumping, and the addition of p H5 in the water phase containing nucleic acids. 0 to 5. 5, the final concentration is 0. After 3M's NaOAc or KOAc, sodium ions neutralized negative charges on the nucleic acid phosphate skeleton, promoting hydrophobic compounding of nucleic acids in an acidic environment.
then add 2 to 2. 5 times the volume of ethanol, after a certain period of incubation, can make the nucleic acids effectively precipitated. Other organic solvents (isopropyl alcohol, polyethyl glycol (PEG), etc.) and salts (10.0mol/L ammonium acetate, 8.0mol/L lithium chloride, magnesium chloride and low concentrations of zinc chloride, etc.) are also used for the precipitation of nucleic acids. Different ions have inhibitory effects on some enzymes or can affect the precipitation and dissolution of nucleic acids, which should be selected when actually used. After centrifugal collection, nucleic acid precipitation with 70% ethanol rinse to remove excess salts, you can obtain purified nucleic acids.
(2) The method of stratification is a separation analysis method based on the differences of some physical and chemical properties of different substances. Including adsorption of layering, affinity layering, ion exchange layering and other methods, including layering methods. It is widely used in the purification of nucleic acids because of the synchronization of separation and purification, and the supply of commodity kits. In certain ion environments, nucleic acids can be selectively adsorbed to the surface of silicone, silicone or glass and separated from other biological molecules. Other selective adsorption methods use modified or wrapped beads as solid-phase carriers, which can be separated by magnetic fields without centrifugation, and nucleic acids combined to solid-phase carriers can be washed away with low-salt buffers or water. The method separates purified nucleic acids and has the advantages of good quality, high yield, low cost, fast, simple, labor-saving and easy automation. glass powder or glass beads have been shown to be an effective nucleic acid adsorbent. In high-salt solutions, nucleic acids can be adsorbed to the glass substrate, and sodium iodide or sodium perchlorate promotes the binding of DNA to the glass substrate. Dederich and others use acid-washed glass beads to isolate purified nucleic acids to obtain high-yielding granulated DNA. In this method, the cells cleavage in alkaline environment, the lysate with potassium acetate buffer neutral, directly added to the glass bead filter containing isopropyl alcohol, isopropanol precipitated particle DNA combined to the glass beads, with 80% ethanol vacuum wash to remove cell fragments and protein precipitation. Finally, DNA combined with glass beads is washed away with a TE buffer containing RNase A, and the resulting DNA can be used directly for sequencing. . Elkin, etc. uses carcification beads to isolate purified granulated DNA. After cell cleavage, the method centrifugally separates the water phase containing the primer, then adds the magnetochemized magnet, and then precipitates it with PEG/NaCl, so that the intended DNA is absorbed to the beads, and finally the magnetic field is isolated from the absorbed DNA, washed by ethanol, washed with water, and a high yield of template DNA applicable to capillary sequencing can be obtained. have also been reported to have purified granule DNA with iron particles as solid-phase supports, isolated by magnetic fields. Bacteria with lysase 2 boiling method lysis, the particles are released into the suspension, iron beads are captured, iron beads are separated by magnetic field, after rinsing and washing the granules with water, high yield, sequencing grade of granule DNA can be obtained. affinity is a type of layering method that separates substances by using the specific affinity between the substances to be separated and their specific ligands. Chandler and others have reported a method of separating nucleic acids using peptide nucleic acids (PNA). PNA is a class of DNA analogas with N-(2-aminoethylene) 2 glycine structural units as skeletons that can be used as reagents for purifying Pic (pg) grade ribosome DNA (rDNA) and ribosome RNA (rRNA). In this method, the biotin-labeled peptide nucleic acids (peptide nucleic acids, PNAs) are used as probes to wrap the magnetic beads of anti-bioprotein streptococcus as solid-phase carriers. PNA probe in a high-salt environment, mixed with the purpose of nucleic acid (DNA or RNA), after boiling, ice bath, temperature-breeding hybridization steps, directly added to the package of anti-bioprotein streptococcus submagnetic particles, by static capture of PNA-nucleic acid hybrid, washing and obtain purified nucleic acid. . Schluep and others also use a tri-helix DNA to isolate prosurbody DNA based on affinity and layering principles. Tri-helix DNA consists of homogeneity 2 homogeneity double helix chain and homogeneity single chain, T identification A on a single chain. T base, pair formation T . A . . . T triplet, protonized monocytosine (C-plus) identifies G. C Base pairs form C . G. C Triple. Under the appropriate conditions, the combination of the three helixes has high specificity and high stability. The lipolymer polysacin oligonucleotide chain is chemically connected to the Sephacryl S21000 SF particles to form affinity vectors. When the proton DNA solution containing the destination sequence is mixed with it, in an acidic environment (p H 4. 5 to 5. 5), the prosurparticles are combined to the affinity carrier particles, and the high concentration of NaCl in the solution stabilizes the triumthlink form and reduces non-specific binding to protein and cell DNA. After a period of reaction, the particle suspension is added to a layer of column, with appropriate scavenging liquid to change the p H value to alkaline environment, can make triple disintegration, mass particles are washed away. By separating the prostrual DNA by this method, the production of the prosury can reach 62% of the added amount. have also been reported using Schizophyllan (SPG) to prepare affinity and layering columns to separate purified RNA. SPG is a beta-21,32 glucosaccharide that, at low temperatures, the flow phase containing RNA is absorbed on the column through a layered column, Poly (C) and poly (A) and SPG through hydrogen bonds and hydrophobic action to form a complex, and then by changing the buffer composition, the adsorbed RNA is washed away. Another example of affinity layering applied to nucleic acid separation and purification is the separation of mRNAs with poly (A) tails from the total RNA of the myumber cells using oligo (dT)2 cellulose layering.
in this method, short-chain olgo (dT) is connected to the cellulose medium by combining its 52 phosphoric acid with the hydroxycodricity of cellulose. As the sample passes through the oligo (dT) column, mRNA is connected to the cellulose medium to separate from other RNAs because it poly (A) forms a stable RNA2DNA hybrid chain with the short chain olgo (dT). Poly (A) RNA can be washed away and purified under suitable conditions (low salt, heating). ion exchange layer is a fixed phase of a substance with ion exchange properties, which can be reversed with the ion energy in the flow phase, thus separating the ionized compounds. The purified nucleic acid is purified by ion exchange layering because nucleic acid is a linear polyanthion with high negative charge, and in the low ion strength buffer, the electrostatic reaction between the functional substituts on the purpose nucleic acid and the anion exchange column is used to bind the negatively charged nucleic acid to the positively charged substitut, and the impurities molecules are purified.
then increase the ion strength of the buffer, the nucleic acids are purified from the substitin, and the purified nucleic acids can be obtained by isopropanol or ethanol precipitation. This method applies to the purification of large-scale nucleic acids. Ferreira, etc. with 0. 5 M NaCl's TE buffer balances the laminate column, and after the sample is sampled, the nucleic acid is washed off with a TE buffer containing 1 M NaCl, resulting in a good separation effect.
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