DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and two optional steps in a DNA extraction:
- Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methods-blending, grinding or sonicating the sample.
- Removing membrane lipids by adding a detergent or surfactants.
- Removing proteins by adding a protease (optional but almost always done).
- Removing RNA by adding an RNase (often done).
- Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt. See ethanol precipitation.
Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg2+ and Ca2+, which prevents enzymes like DNase from degrading the DNA.
Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation.
If desired, the DNA can be reDNA isolation methods
Many different methods and technologies are available for the isolation of genomic DNA. In general, all methods involve disruption and lysis of the starting material followed by the removal of proteinsand other contaminants and finally recovery of the DNA. Removal of proteins is typically achieved by digestion with proteinase K, followed by salting-out, organic extraction, or binding of the DNA to a solid-phase support (either anion-exchange or silica technology).
DNA is usually recovered by precipitation using ethanol or isopropanol. The choice of a method depends on many factors: the required quantity and molecular weight of the DNA, the purit required for downstream applications, and the time and expense.
The separation of DNA from cellular components can be divided into
four stages:.
- Disruption
- Lysis
- Removal of proteins and contaminants
- Recovery of DNA
In some methods, stages 1 and 2 are combined.solubilized in a slightly alkaline buffer or in ultra-pure water.
