Molecular diagnosis uses nucleic acid as the detection object and is widely used in the clinical diagnosis of tumor, infection, etc. Most importantly, nucleic acid (DNA & RNA) extraction is the “first step” of molecular diagnosis. By coating the magnetic beads (MBs) with some functional groups lick silicon, amino, carboxyl, etc., high-throughput and automated extraction process of nucleic acids can be achieved. This technology was developed in the 1980s, and now there are mature kits and formed an industry.
Steps and Principles
Fig. 1 Schematic diagram of the steps of MBs-based extraction of nucleic acids.
MBs-based nucleic acid extraction can generally be divided into four steps: lysis-binding-washing-elution. First of all, the nucleic acids (DNA/RNA) in the cell or tissue is released under the action of the lysis solution. Then, the binding of nucleic acids to MBs mainly depends on electrostatic interaction, hydrophobic interaction and hydrogen bonding. In the reaction system of the MBs-based extraction, nucleic acid molecules will be compressed from linear to spherical shape, exposing a large number of negatively charged groups on the nucleic acid backbone to connect with the cations in the reaction system. Under the action of the negatively charged groups on the outermost layer of the MBs, a salt bridge structure of “anion-cation-anion” is formed, so that nucleic acid molecules are specifically adsorbed to the surface of the MBs. After the reaction buffer is discarded, adding water molecules will quickly and fully hydrate the nucleic acid molecules, thus the ionic interaction is removed and the nucleic acid molecules adsorbed on the MBs are purified.
Advantages of MBs-based Nucleic Acids Extraction
The MBs-based nucleic acid extraction is a perfect combination of nanotechnology and biotechnology, and has incomparable advantages over other extraction methods:
- Low–demand sample: a high concentration of nucleic acids can be obtained using a small amount of material.
- Simple and fast operation: the entire operation process is basically divided into five steps (lysis, binding, washing, drying, elution), the whole process does not require centrifugation, most of the steps can be completed within 30 ~ 60 minutes.
- Stable and reliable quality: free MBs can bind more nucleic acids, and the specific binding makes the purity of nucleic acids higher. The recovery rate can be adjusted and improved by controlling the surface groups of MBs.
- Fully automatic operation: the use of nucleic acids extractor can realize automatic and high-throughput operation, as well as can realize the extraction of tens or even hundreds of samples.
- Safety and non-toxicity: The reagents do not contain toxic chemical reagents such as phenol and chloroform, which are fully in line with modern environmental protection concepts.
Magnetic beads-based nucleic acids extraction has been widely used in molecular biology, such as genome research, molecular evolution research, medical genetic disease research, mutation gene detection, tumor screening, HPV detection, HLA typing, transplant matching, etc. Moreover, it has been applied in the detection of forensic biological samples like blood spots, sperm spots, hair, cigarette butts and other on-site evidence, providing evidence for judicial paternity testing.
Precautions for Use
- More MBs may not lead to better extraction effect
When the extraction effect is not good, it is not advisable to increase the amount of MBs.
The main feature of MBs is that they can be uniformly dispersed in the liquid and can be separated from the liquid phase in a solid state under the action of an external magnetic field. For any reagent system, the ratio of MBs to the liquid should have a certain threshold. Excessive MBs will lose their dispersion characteristics because they cannot be uniformly dispersed in the liquid, and the contact efficiency between the nucleic acid magnetic beads and the liquid cannot be fully increased during the washing process. Excessive MBs will also adsorb more impurities, which has a great influence on the effect of impurity removal. Even sometimes, too much MBs will adsorb protease, lysozyme and other functional components that play a major role in the liquid system, resulting in a lower efficiency of the entire extraction process.
- More reagents may not lead to better extraction effect
For the MBs method, each increase in the volume of the liquid will reduce the probability of MBs collisions, further resulting in a significant drop in the adsorption rate. Therefore, in many cases, although adding lysis solution and/or washing solution can indeed enhance the lysis and washing effect, the key point of MBs extraction is the efficiency of MBs adsorption of nucleic acids. And if the MBs collision efficiency cannot be guaranteed, neither do the nucleic acids extraction efficiency. Therefore, simply increasing the amounts of reagents (lysis solution and/or washing solution) used to improve the extraction effect may not be completely effective.
- Longer shing times may not lead to better extraction effect
When there are too many impurities in the extracted nucleic acids, users will consider washing several more times to obtain more pure nucleic acids. Increasing washing times is indeed conducive to the purification, but considering that each wash will lose a certain amount of nucleic acids and increase the possibility of fragmentation and hydrolysis, it is generally appropriate to control the number of washes at 2 ~ 4 times.
- More samples may not lead to the better extraction effect
When the sample is not fresh enough or the nucleic acids content is small, the nucleic acids extraction effect is often not good. In this case, many users will use multiple samples to increase the amount of extracted nucleic acids. However, simply increasing the sampling volume can sometimes introduce too many impurities, exceeding the lysing capacity and also reducing the extraction efficiency. Therefore, it is not recommended to simply increase the sampling volume to obtain more extraction volume.
If the extraction volume is indeed too low due to insufficient sampling volume, it is recommended to go through an enrichment or concentration step before starting the extraction, or to increase the completeness of lysis and expose more nucleic acids.
- One certain kind of MBs is not versatile for all tests
There are many types of MBs, and they have different particle sizes, different dispersions, different magnetic response times, different matrixes, different outer modified functional groups and different coating densities, etc., and all of which will lead to great difference in MBs features. Therefore, the experiments and systems that different magnetic beads adapted to are also different. Some MBs show higher adsorption efficiency in the extraction of constant nucleic acids, while some MBs are more suitable for the extraction of trace amounts of nucleic acids. Some MBs are suitable for acidic reagent systems, while some MBs are suitable for alkaline reagent systems. Some magnetic beads have good magnetic responsiveness but fast sedimentation speed, which are more suitable for magnetic rod type automatic extractor; on the other hand, some magnetic beads have slow sedimentation speed but long magnetic response time, which are more suitable for pipette type automatic extractor. In most cases, except for the fixed kit, the choice of MBs and reagent system need to be adjusted for certain needs.
1. Saiyed, Z. M.; Ramchand, C. N.; et al. Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support. Journal of Physics: Condensed Matter 2008, 20 (20), 204153.
2. Boom, R.; Sol, C. J.; et al. Rapid and simple method for purification of nucleic acids. Journal of clinical microbiology 1990, 28 (3), 495-503.
3. Berensmeier, S., Magnetic particles for the separation and purification of nucleic acids. Applied microbiology and biotechnology 2006, 73 (3), 495-504.