DNA extraction

• DNA isolation is a process of purification of

DNA from sample using a combination of
physical and chemical methods.
• The first isolation of DNA was done in 1869
by Friedrich Miescher
 Basic procedure 

• 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 which also serves in cell lysis.
• Removing proteins by adding a protease (optional but often
done).
• Removing RNA by adding an RNase (almost always done).
• DNA purification from detergents, proteins, salts and
reagents used during cell lysis step. 
 Most commonly used procedures for DNA
purification
• Ethanol precipitation
• Phenol–chloroform extraction 
• Minicolumn purification
 Ethanol precipitation
•  Done by ice-cold ethanol or isopropanol.
Since DNA is insoluble in these alcohols, it will
aggregate together, giving a pellet upon
centrifugation. Precipitation of DNA is
improved by increasing of ionic strength,
usually by adding sodium acetate.
 Phenol–chloroform extraction
•  In which phenol denatures proteins in the
sample. After centrifugation of the sample,
denaturated proteins stay in organic phase
while aqueous phase containing nucleic acid is
mixed with the chloroform that removes
phenol residues from solution.
• (Note: for DNA isolation in used phenol
buffered to pH 8, RNA must be isolated using
acidic phenol.)
 Minicolumn purification
•  It relies on the fact that the nucleic acid may
bind (adsorption) to the solid phase (silica or
other) depending on the pH and the salt
content of the buffer.
• 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.
• After isolation, the DNA is dissolved in slightly alkaline
buffer, usually in the TE buffer, or in ultra-pure water.
 Detecting DNA

• A diphenylamine (DPA) indicator will confirm the presence of

DNA.
• This procedure involves chemical hydrolysis of DNA: when
heated (e.g. ≥95 °C) 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.