DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic steps in a DNA extraction:
- Breaking the cells open, commonly referred to as cell disruption, to expose the DNA within, such as by grinding or sonicating the sample.
- Removing membrane lipids by adding a detergent.
- Precipitating the DNA with an alcohol — usually ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet on centrifugation. This step also removes alcohol-soluble salt.
Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg2+ and Ca2+. This stops dnase enzymes from degrading the DNA.
Cellular and histone proteins bound to the DNA can be removed prior to its precipitation either by adding a protease or having prior to precipitation, precipitating with sodium or ammonium acetate, or extracting with a phenol-chloroform mixture.
If desired, the DNA can be resolubilized in a slightly alkaline buffer.
A diphenylamine (DPA) indicators will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95oC) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the 2-deoxyribose is converted to w-hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations.
Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nm, and aromatic proteins absorbs UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.
DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an agarose gel, staining with ethidium bromide or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration.
Source: Wikipedia (All text is available under the terms of the Creative Commons Attribution-ShareAlike License)