Views: 0 Author: BunnyTeeth Technology Inc. Publish Time: 2022-05-20 Origin: BunnyTeeth Technology Inc.
The Magic Ball of Biology - Magnetic Beads
1. The discovery of magnetic beads
The idea for the invention of magnetic beads originally came from John Ugelstad, a chemist at the Norwegian University of Science and Technology, who in 1976 produced uniformly magnetized spherical particles using polystyrene as the main material.
In 1979 Vogelstein et al. reported the use of glass powder as an adsorbent can be used to extract DNA fragments from sepharose in the presence of high concentrations of sodium iodide, and later solid-phase nucleic acid purification techniques based on silica gel and other surface carriers with hydrophilic properties were widely developed. The magnetic particle-based nucleic acid purification method is one of them.
2. Types of magnetic beads
Basic structure (generally divided into 3 layers):
The innermost layer is polystyrene (PS).
The second layer is wrapped with a magnetic substance (usually Fe3O4).
The outermost layer is a polymer modified by functional groups (such as carboxyl-COOH, hydroxyl-OH), which can be combined with nucleic acid.
Particle size: different sizes of magnetic beads have obvious suspension differences. The smaller the size of suspension, the better, but the magnetic response will be weakened. The smaller the size, the better the suspension, but the magnetic response will be weakened. Generally, for small samples with low nucleic acid content, it is better to use magnetic beads with good suspension.
Surface modification (functional group): -OH/-COOH/-NH4/Protein
Difference in use (environment of action): both -OH and -COOH magnetic beads can adsorb nucleic acids effectively. Generally speaking, beads with hydroxyl are relatively better for nucleic acid adsorption in chaotropic salt system; beads with carboxyl function group are relatively better for DNA and RNA adsorption in PEG system.
Magnetic beads Buffer: generally organic solvents such as polyethylene glycol (PEG).
3. Principle of magnetic bead method
SPRI (Solid Phase Reverse Immobilization) is that paramagnetic beads can selectively bind to nucleic acids by type and size, and can be used to isolate, purify and clean up high purity nucleic acids. This technology can be applied to next generation sequencing (NGS), Sanger sequencing, qPCR, ddPCR and microarrays, etc.
Based on SPRI technology, in the magnetic bead method reaction system, nucleic acid molecules (DNA & RNA) will be compressed from linear to spherical shape. DNA will undergo dramatic changes in DNA molecular conformation promoted by NaCl or MgCl2 in the presence of certain concentration of PEG. A large number of negatively charged phosphate groups on the phosphate backbone will bind to the negatively charged carboxyl magnetic beads on the surface. This interaction between negative and negative charges is currently thought to be due to the action of positively charged salt ions (e.g., Na+). The negatively charged phosphate group forms an "anion-cation-anion" salt bridge structure with the carboxyl group through the dissociated salt ion (e.g. Na+), so that DNA is specifically adsorbed to the surface of the carboxyl beads. When the reaction buffer is discarded, the addition of water molecule rapidly and fully hydrates the nucleic acid molecules and releases the ionic interaction between the three, allowing the purification of the nucleic acid molecules adsorbed to the magnetic beads. Using the magnetic properties of the magnetic beads, DNA fragments can be collected and eluted by an applied magnetic field.
- The longer the DNA molecule, the more negatively charged phosphate groups are exposed on its surface, the more negatively charged the whole molecule is, and it is easier to be adsorbed onto the surface of the magnetic beads so only lower concentrations of PEG and NaCl are required for recovery.
- The shorter the DNA molecule is, the higher the concentration of PEG and NaCl is needed to destroy the hydrated layer on its surface more thoroughly and expose enough negatively charged phosphate groups to be adsorbed and recovered by the magnetic beads. (i.e. the smaller the recovered DNA fragment, the higher the concentration of PEG and NaCl that needs to be added and the larger the volume of magnetic beads.)