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DNA Extraction and Quantitation for Forensic Analysts

Removal of Denatured Proteins

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Step Three: Removal of Denatured Proteins

Photo of compounds used for removal of denatured proteins
National Institute of Justice (NIJ) (see reuse policy).

Denatured proteins are removed during the extraction using a phenol chloroform isoamyl alcohol (PCI) combination. Phenol denatures proteins that are subsequently hydrolyzed with Proteinase K. Due to their negative charge, DNA molecules can be separated from other cellular components. Addition of PCI to a sample promotes separation of non-polar (organic) and polar (aqueous) phases. The phenol is not miscible with water, denatures protein, and sequesters the denatured hydrolyzed protein in the organic phase. During this process, the DNA remains in the aqueous phase in its double-stranded state.

PCI may be purchased commercially or prepared in a ratio of 25:24:1. Because the pH of phenol (approximately 7.0) would generally be too acidic for purposes of DNA extraction, the phenol is buffered by saturation with TE buffer.

Care must be taken to isolate only the aqueous phase during this procedure. Any residual PCI can compromise the filters in the filtration devices and also acts as a PCR inhibitor.
Diagram of chemical makeup of Phenol
National Institute of Justice (NIJ) (see reuse policy).

Step Three Reagents

  • Phenol

Phenol is a member of a class of organic compounds containing a hydroxyl group fixed to an unsaturated carbon in a benzene ring. Phenols are not true alcohols; they are more acidic than alcohols but less so than carboxylic acids. In most reactions they behave as nucleophiles. Phenols are also readily oxidized, more so than alcohols. This property is employed in DNA extraction. The addition of hydroxyquinoline to the reagent gives the organic phase an orange color, making it easier to differentiate the aqueous phase containing DNA. Polysaccharides and proteins are soluble in phenol, allowing for their separation from DNA.

Safety Note: CAS# 108-95-2
Although originally used for its antiseptic properties, phenol is highly toxic and should be handled in a fume hood while wearing personal protective equipment. Skin contact and inhalation should be avoided.
Diagram of chemical makeup of Chloroform
National Institute of Justice (NIJ) (see reuse policy).
  • Chloroform

Chloroform (CHCl 3), or trichloro-methane, is a colorless liquid that is slightly water-soluble and miscible with organic solvents such as phenol. It is more dense than water or buffer (in which DNA is soluble), yet less dense than phenol. As it increases the phenol phase density, it promotes a sharp interface between the organic and aqueous layers. Chloroform also solubilizes lipids. During the extraction procedure, cellular debris that is not totally digested can be observed at the interface.

Safety Note: CAS# 67-66-3
Previously widely used as an anesthetic, inhalation of chloroform depresses the central nervous system. It is also a suspected teratogen and known carcinogen and should never be handled outside of a fume hood.
Diagram of chemical makeup of Isoamyl Alcohol
National Institute of Justice (NIJ) (see reuse policy).
  • Isoamyl Alcohol

Isoamyl alcohol, or 3-methyl-1-butanol, is a primary alcohol. A liquid solvent, it is often included in genomic extraction protocols to help prevent foaming of the reagents, making it easier to detect the interface between the organic and aqueous phases. It is included in the protocol in very small concentration, compared to the other reagents.06

Safety Note: CAS# 123-51-3
A component of some paint strippers and other solvents, isoamyl alcohol causes irritation upon skin contact or inhalation. Vapors can also cause ocular discomfort and effects. This reagent should be handled with care and in a fume hood.

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