Article source: Luoyang Jiente Biotechnology Co., Ltd.
Magnetic beads extraction is a novel nucleic acid extraction technology based on nano-biomagnetic beads. The nucleic acid molecules can specifically recognize and combine with the silicon hydroxyl groups on the surface of the magnetic beads, and aggregate or disperse under the action of external magnetic fields. Eliminate the manual process of centrifugation, extraction of supernatant, etc. in the traditional nucleic acid extraction process, thus achieving automatic extraction of nucleic acids. Widely used in clinical disease diagnosis, blood transfusion safety, forensic identification, environmental microbial detection, food safety testing, molecular biology research and other fields.
Magnetic bead nucleic acid extraction generally includes four main steps of cleavage, binding, washing and elution, each step can be realized by a plurality of different methods individually or in combination, wherein the cleavage step is the most important to determine the quality of the extraction, often Some teachers will also ask how to optimize the lysate. So, let's analyze the reagents that are often used to configure lysates and how they work.
1 , guanidine hydrochloride, urea
Hydroquinone can break the hydrogen bond at a concentration of 4-8 mol. There are two possible mechanisms: 1. The denatured protein and the guanidine hydrochloride and urea preferentially combine to form a denatured protein-denaturing agent complex. When the complex is removed, the ND reaction is caused. The balance moves to the right. As the concentration of the denaturant increases, the protein in the natural state continuously transforms into a complex, which eventually leads to complete denaturation of the protein. 2. The guanidine hydrochloride and urea have a solubilizing effect on the amino acid and can form a hydrogen bond. When the concentration is high, the concentration is high. It can destroy the hydrogen bond structure of water and become a better solvent for non-polar residues, so that the hydrophobic residues inside the protein can be stretched and dissolved. The protein denaturation caused by guanidine hydrochloride and urea is often irreversible.
At the same time, guanidine hydrochloride is a strong inhibitor of nucleases, but it is not a sufficiently strong denaturant to allow intact RNA to be extracted from RNase-rich tissues. High concentrations of urea can also denature proteins and inhibit Rnase activity.
2 , guanidinium isothiocyanate
A protein denaturant that rapidly dissolves proteins, causing cell structure to break down, and nuclear proteins are rapidly separated from nucleic acids due to the disappearance of their secondary structure. The most potent of the commonly used protein denaturants are guanidinium isothiocyanate, which converts most proteins into a random coiled state. Contains strong anionic and cationic groups that form strong hydrogen bonds. In the presence of a reducing agent, guanidinium isothiocyanate can break the hydrogen bond, and in the presence of a detergent such as SDS, the hydrophobic action can be disrupted.
3 , dithiothreitol ( DTT )
The main function of dithiothreitol is to destroy the disulfide bond in the RNase protein, denature the Rna enzyme, and inhibit the cleavage of the phosphodiester bond caused by phenol oxidation and lead to the crosslinking of RNA and DNA.
Dithiothreitol has a low pungent odor and low toxicity. DTT is less stable due to its tendency to be oxidized by air; however, cryopreservation or treatment in an inert gas can extend its useful life. Due to the low nucleophilicity of protonated sulfur, the effective reduction of DTT decreases with decreasing pH; DTT or DTT-containing solutions cannot be subjected to high pressure treatment.
4 , proteinase K
A serine protease with a broad cleavage activity. It cleaves the carboxy terminal peptide bond of an aliphatic amino acid and an aromatic amino acid. This enzyme was purified to remove RNase and DNase activity. Since proteinase K is stable in urea and SDS and has the ability to degrade natural proteins, it is widely used, including preparation of chromosomal DNA for pulse electrophoresis, Western blotting, and removal of nucleases in DNA and RNA preparation. The working concentration of proteinase K is 50-100 μg/ml. It is active in a wide pH range (pH 4-12.5).
The above are commonly used reagents in lysates. In practice, different concentrations of lysis buffer should be prepared for different samples (plant tissue, animal tissue, whole blood, serum, plasmid), and according to the state (mass, volume) of the sample. And the desired experimental results (nucleic acid concentration, OD ratio) adjust the concentration of the lysate, in addition, the three steps of binding, washing and elution are optimized to obtain a perfect nucleic acid extraction scheme.
Teachers have any questions about the magnetic bead nucleic acid extraction program. Welcome to the official website of Giant-bio for discussion.
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