DNA-isolatie

DNA needs to be isolated from cells and cut at precise positions for many applications, such as recombinant DNA technology.

Though different types of DNA extraction methods are used for different cell types, there are three standard steps: cell lysis, protein removal, and DNA recovery.

Inside eukaryotic cells, DNA is packed within the nucleus. Thus, both the cell membrane and the nuclear membrane need to be ruptured to isolate the DNA.

This step can be done mechanically by breaking down cells through grinding or sonication, or chemically by using detergents and enzymes to dissolve parts of the cell membrane.

Once the cell contents are released, the debris is separated from soluble components by centrifugation. The supernatant recovered contains nucleic acids and water-soluble proteins.

To remove proteins, enzymes, such as proteinase K, peptidase, or lysozyme, are added to the supernatant to break the peptide bonds.

DNA is recovered from the supernatant through precipitation by adding alcohol and a salt, such as sodium acetate.

The isolated precipitate is dissolved in water or a buffer.