- Lysis
The first step is breaking down the nucleus and the cell to release the DNA. With the help of mechanical disruption, medical scientists break open the human cell. They cut the tissue into small pieces. They can also use a small blender, mortar, pestle, and tissue homogenizer. The plant cells have tough cell walls. Enzymes like Proteinase K release cellular proteins and DNA on softer cells.
- Precipitation
After the Lysis, the DNA is free from the nucleus but it is still in mixture with the cell parts. This process frees the DNA from the cellular components. The DNA molecules have negative charges, which undergo neutralization by sodium ions, making them more soluble. However, their water solubility decreases. Adding alcohol like isopropanol or ethanol causes the DNA to precipitate out of the aqueous solution as it is insoluble in alcohol.
- Purification
Precipitation has separated the DNA from the aqueous phase. Rinse it with alcohol to remove the cellular debris. Then redissolve the purified DNA with water for storage and handle it easily.
- Lysate Creation
All three methods- physical, chemical and enzymatic- are used for lysate creation. In the physical method, the lysate is made from tough tissues. The scientist will freeze the cells and grind them with the mortar and pestle under liquid nitrogen. Then they are exposed to enzymatic or chemical lysis conditions.
- Lysate Clearing
The Cell-Free Nucleic Acid Extraction Kit can help to carry out this process. The unwanted materials of the cells, like the lipids, saccharides, and proteins from the cell itself, can clog the membranes and interfere with the other applications of the purification process. Therefore, scientists need to establish a clearing or filtration or centrifugation.
Centrifugation is the most suitable method to clear out the huge pile of debris. However, it is a lengthy process. Filtration, on the other hand, is a faster method, but a large amount of debris may clog the membrane. Automated protocols can use bead-based clearing but are usually overwhelmed with biomass. After the clearing, the DNA goes through a purification process.
