In-vitro diagnosis refers to products and services that obtain clinical diagnostic information by testing human samples (blood, body fluids, tissues, etc.) outside the human body to determine diseases or body functions.
One of the key steps in the detection of human samples is the purification of nucleic acids (DNA and RNA) in the sample. The method of nucleic acid purification is an important factor affecting the quality of the extracted nucleic acid, and only high-quality nucleic acid can meet various downstream applications.
Nucleic acid is the basis of molecular biology, and nucleic acid extraction is a threshold for nucleic acid detection, and even the entire molecular industry cannot bypass the threshold. In many cases, the quality of nucleic acid extraction from a sample directly determines the validity of the test results.
Basic knowledge of nucleic acid
Nucleic acid is divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), among which RNA can be divided into ribosomal RNA (r RNA), messenger RNA (m RNA) and transfer RNA (t RNA) according to different functions.
DNA is mainly concentrated in the nucleus, mitochondria and chloroplasts, while RNA is mainly distributed in the cytoplasm.
Why is nucleic acid extraction needed
Nucleic acid extraction provides answers to a large number of extensive research and applications, and the obtained nucleic acid can be used in a variety of ways. The exact research purpose determines the type of nucleic acid to be extracted; the application of nucleic acid often affects the choice of extraction method. In order to determine the best research method, it is necessary to have a clear understanding of the downstream applications of nucleic acids and any potential limitations related to the type of sample. Although the method of cell lysis is different depending on the sample type, the core principle of the overall nucleic acid extraction remains the same: cell or tissue samples are lysed to remove non-nucleic acid contaminants.
Principles and requirements of nucleic acid extraction and purification
1. Ensure the integrity of the primary structure of nucleic acid;
2. Eliminate pollution from other molecules (such as eliminating RNA interference when extracting DNA);
3. There are no organic solvents and excessively high concentrations of metal ions that can inhibit enzymes in nucleic acid samples;
4. The pollution of other biological macromolecules such as proteins, polysaccharides and lipid molecules should be minimized;
5. Eliminate the contamination of other nucleic acid molecules, such as removing RNA when extracting DNA molecules, and vice versa.
Common nucleic acid extraction and purification methods
1. Phenol and chloroform extraction method
Phenol and chloroform are used to extract DNA by using phenol as a protein denaturant. Repeated extraction will denature the protein. SDS (sodium dodecyl sulfonate) will lyse the cell membrane and digest the protein or polypeptide or small peptide in the presence of proteinase K and EDTA. Molecules, denature and degrade the nuclear protein, freeing DNA from the nuclear protein. While DNA is easily soluble in water, but insoluble in organic solvents. The surface of protein molecules has hydrophilic groups, which are also easy for hydration, and a hydration layer is formed on the surface, so that the protein molecules can smoothly enter the aqueous solution to form a stable colloidal solution.
When the organic solution exists, the colloidal stability of the protein is destroyed, denatured and precipitated. After centrifugation, the organic solvent is in the bottom of the test tube (organic phase), the DNA is in the upper aqueous phase, and the protein is precipitated between the two phases. Using the extraction principle, according to the protein nucleic acid dissolved in different reagent layers, the pipette tip is extended into the different liquid layers to extract the required components, and purified nucleic acid is obtained after multiple washings.
The disadvantage is that due to the use of phenol, chloroform and other reagents, the toxicity is relatively high, long-term operation has a greater impact on personnel health, and the recovery rate of nucleic acid is low, and the loss is large. Due to the large operating system, the operation repeatability of different experimenters is poor. , It is not conducive to protecting RNA, and it is difficult to carry out miniaturization operations.
The advantage is that it uses common reagents and medicines in laboratories, and the cost is relatively low.
2. Spin column purification
This method utilizes the surface of nucleic acid to be covered with a thin film composed of water molecules. In a high-salt environment, this hydrophilic membrane will be destroyed so that nucleic acid can be adsorbed on the spin column, while other impurities such as protein and metabolism Products etc. will be separated from nucleic acid by centrifugal precipitation.
But even so, the spin column purification method still has shortcomings:
(1) Highly dependent on the binding capacity of the adsorption membrane;
(2) Incomplete elution leads to the loss of nucleic acid;
(3) Nucleic acid cannot be extracted in large quantities.
3. Magnetic bead method for nucleic acid separation technology
The positively charged magnetic beads are easy to adsorb negatively charged nucleic acids and are mainly used to extract DNA and RNA from human serum or plasma samples. Generally speaking, the sample is mixed with the binding buffer and magnetic beads. After the nucleic acid is combined with the magnetic beads, it is washed and captured by the magnetic field several times. The eluted nucleic acid can be detected by PCR or other specific methods to detect specific DNA or RNA. .
Magnetic separation is also widely used in the diagnostic industry, and can achieve high-throughput and automated nucleic acid extraction methods.
Different types of genetic testing will involve nucleic acid extraction and purification steps. Among the methods of nucleic acid extraction and purification, both the liquid method and the spin column method involve the use of toxic organic substances, which are harmful to the environment and experiment operators.
The magnetic bead method requires corresponding specially formulated magnetic bead buffer and specially modified magnetic beads. From the cost and operation aspects, both have high cost and cumbersome operation steps. At the same time, it also limits the automation process and the promotion of applications. . Therefore, the development of a rapid nucleic acid release method that can quickly release nucleic acid without affecting subsequent genetic testing experiments has become an urgent problem to be solved.
4. Gene nucleic acid rapid release technology
The DNA release kit is dedicated to quickly release constant temperature fluorescence amplified DNA from various common samples. It has the following characteristics:
1. The nucleic acid release agent can quickly lyse the sample and free the DNA of the sample in the solution. There is no need to prepare any reagents by yourself, and no toxic reagents such as phenol and chloroform are used.
2. The operation is simple, it only takes one step (about 3 minutes) to obtain the DNA template for constant temperature fluorescence amplification, without any operation steps such as centrifugation and extraction.
3. The whole process is only completed in one centrifuge tube, which avoids the loss of trace samples and is not easy to cross contamination. It is simpler and more convenient than commonly used extraction kits.
4. Wide compatibility, suitable for common molecular biology samples, including bacteria, insects, various animal tissues, oral cells, hair, bacteria and viruses in tissues, etc.