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DNA and RNA remain in water layer

Centrifuge

Proteins now in phenol layer _

DNA and RNA remain in water layer

Centrifuge

Proteins now in phenol layer _

FIGURE 21.01 Phenol Extraction Removes Proteins from Nucleic Acids

Proteins can be removed from a solution of DNA or RNA by adding an equal volume of phenol. The phenol dissolves the proteins without disrupting the DNA or RNA. Since phenol is very dense, it forms a separate layer at the bottom of the tube. When the two solutions are shaken, the proteins dissolve into the phenol. The two layers separate again after a brief spin in the centrifuge. The top phase, which now contains just DNA and RNA, can be isolated.

Nucleic acids may be purified on columns containing resins that bind DNA and RNA.

When phenol is added to water, the two liquids do not mix to form a single solution; instead, the denser phenol forms a separate layer below the water. When shaken, the two layers mix temporarily, and the proteins dissolve in the phenol. When the shaking stops, the DNA solution and phenol containing the proteins separate into two layers (Fig. 21.01). To ensure that no phenol is trapped with the DNA, the sample is centrifuged briefly. Then the water containing the DNA and RNA is sucked off and kept. Generally, several successive phenol extractions are performed to purify away the proteins from DNA.

A variety of newer techniques have been developed that avoid phenol extraction. Most of these involve purifying DNA by passing it through a column containing a resin that binds DNA but not other cell components. The two main choices are silica and anion exchange resins. Silica resins bind nucleic acids rapidly and specifically at low pH and high salt concentrations. The nucleic acids are released at higher pH and low salt concentration. Anion exchange resins, such as diethylaminoethyl-cellulose, are positively charged and bind DNA via its negatively charged phosphate groups. In this case binding occurs at low salt concentrations and the nucleic acids are eluted by high concentrations of salt, which disrupt the ionic bonding.

Unwanted RNA is often removed by degrading it enzymatically.

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