At present, the most effective method for animal molecular research is from the level of nucleic acid molecules, mainly for the identification of species, the origin of species, the assessment of diversity, the study of kinship, system evolution, etc. Due to the limitations of field extraction, many materials must be processed and brought back to the laboratory for DNA extraction. In addition to the following methods, and there are other methods such as: DNA extractor, RNA extractor, DNA extraction kit, RNA extraction kit and so on.
1. Classical extraction method
Isopropanol precipitation, phenol extraction, and formazan cleavage are the three most classic methods for extracting DNA. They use proteinase K and sodium lauryl sulfate to digest and disrupt cells.
In the first two methods, the lysate is first used to remove proteins with chloroform or phenol, and then the DNA is precipitated with isopropanol or ethanol, respectively. The methylphthalamide method uses a high concentration of methylphthalamide to depolymerize the binding of protein and DNA, and then dialysis is used to process the DNA sample.
The DNA obtained by these classical methods is of high purity and can meet the requirements of various tests, but the operation is cumbersome and time-consuming, and the reagents used have certain toxicity.
2. Glass rod winding method
The glass rod winding method is to lyse the cells with hydrochloric acid muscle, and then spread the cell lysate on the ethanol in the centrifuge tube. Use a glass rod to slowly stir between the ethanol and the lysate to wind the precipitated colloidal DNA on the glass rod Methods.
This method has high requirements for operation technology, but it is simple and fast.
3. Glass particle adsorption method
The animal cells are lysed with the cell lysate containing muscle isothiocyanate, and then glass particle buffer is added, the glass particles combined with chromosomal DNA are collected by precipitation, and then the eluate is used for elution to obtain DNA.
4. Fast extraction method of blood cell DNA
Use non-ionic denaturant NP40 (ethyl phenyl polyethylene glycol) instead of SDS to lyse the cells, extract the nucleus, and then extract DNA with phenol or chloroform. In this way, the purity of the DNA isolated from the blood is high, which can meet the needs of various clinical tests and experiments. NP40 and Tween-20 can also be used to break the cell membrane at the same time to avoid the negative effect of SDS on the subsequent steps.
5. TLS method
The TLS method is the triethanolamine lauryl sulfate method. TLS is a strong surfactant. First, TLS is directly applied to the cell or tissue homogenate, after dissolving the cell membrane and nuclear membrane, the DNA is extracted, and then the DNA is purified by conventional methods. After TLS is combined with protein, it will lose its binding effect on DNA, so it can completely separate protein from DNA. In addition, the use of TLS method to extract DNA from tissues, cells and peripheral blood can overcome the problem that the protease hydrolysis conditions used in conventional methods are not well mastered.
6. Salting out method
The salting-out method is a method of using saturated sodium chloride to remove protein and obtain DNA. The quality of DNA extracted by this method is acceptable, but the productivity is low, and the purity is poor.
7. Other DNA extraction methods
The method of extracting DNA from pathological specimens usually adopts heating or microwave method to remove embedded paraffin, column chromatography or salting out method to remove protein to extract DNA. This method is relatively safe and simple.
For the specific steps of the extraction of animal genomic DNA, please refer to "Extraction of Total Genomic DNA of Animal Tissues"