Phenol-Chloroform Extraction of Nucleic Acid

  • Phenol-chloroform extraction can be used to isolate and purify nucleic acid (DNA and RNA). It is a very reliable method to remove proteins and lipids from the nucleic acid. However, it is now an outdated method and is replaced by commercially available nucleic acid binding columns and purification kits.
  • It is an example of liquid-liquid extraction which is based on differential solubilities of biomolecules (e.g., nucleic acids, proteins, carbohydrates, and lipids) in water and phenol/chloroform.
  • Since most proteins and lipids are highly soluble in phenol, they move to the organic phase, whereas nucleic acid stays in the aqueous phase due to its high solubility in water.
  • Since carbohydrates prefer to stay in the aqueous phase due to their high solubility in water, this method is not good at removing carbohydrates from the nucleic acid.
  • Phenol has a slightly higher density (1.07 g/cm3) than water, but when chloroform is added which has a comparatively very high density (1.47g/cm3), the density of the phenol-chloroform solution increases considerably. Therefore during extraction, the organic phase is always present at the bottom, allowing the aqueous phase to be transferred efficiently. In situations where samples contain very high salt concentration or high sucrose concentration, the aqueous phase can be formed at the bottom.
  • The solution of phenol and chloroform is not only more efficient at denaturing proteins but also reduces the partitioning of poly(A)+ mRNA into the organic phase.
  • Phenol and chloroform form homogeneous solution due to very high solubility of phenol in chloroform. In contrast, a homogeneous mixture of phenol:chloroform form a sharp interface with water due to their weak solubility in water (phenol solubility in water is 8.03 g/100 ml and Chloroform solubility in water is 1.06 g/100 ml at room temperature) and high density of the solution.
  • Generally a 1:1 ratio of phenol:chloroform is used for the extraction of nucleic acid. Sometimes a small quantity of isoamyl alcohol is added to this mixture (25:24:1 ratio of Phenol: Chloroform: Isoamyl Alcohol). Isoamyl alcohol is an anti-foaming agent, thus prevents form-formation during the extraction process.
  • In this procedure, an approximately equal volume of phenol:chloroform solution is added to the sample (aqueous solution DNA/RNA or cell lysate or tissue lysate) and mixed. When this mixture is centrifuged, the solution is separated into two phases: the upper aqueous phase and the bottom organic phase (Phenol/Chloroform). At the interface, insoluble material which is nothing but the cell debris and denatured proteins is collected. The aqueous phase which contains nucleic acid is then transferred to another fresh vial and subjected to ethanol or isopropanol precipitation to concentrate nucleic acid.
  • Tris saturated phenol (pH ≈8.0) is used for the purification of DNA, whereas water-saturated phenol (pH ≈4.8) is used for the purification of RNA.
  • At a slightly alkaline pH, the phosphate backbone of both DNA and RNA is negatively charged, therefore, they remain in the aqueous phase. As the pH of the Phenol/Chloroform solution drops, DNA tends to move to the organic phase and at pH 4.8 most DNA is present either at interphase (mostly large DNA fragments) or in the organic phase (smaller DNA fragments). The reason why DNA moves to the organic phase, but not RNA is because of the higher pKa value of DNA, which results in the neutralization of the negatively charged phosphate backbone of DNA.
  • Since phenol is slightly soluble in water (≈7-8% in water), the aqueous phase can be re-extracted with chloroform alone to remove traces of phenol from the final preparation of nucleic acid.  Traces of phenol can inhibit downstream enzymatic reactions and interfere with the spectroscopic analysis.

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