Phenol/Chloroform Extraction

and Ethanol Precipitation





Phenol/Chloroform Extraction

One of the most commonly used and useful methods for isolation and concentration of DNA and RNA from aqueous solutions is phenol/chloroform extraction followed by ethanol precipitation. During organic extraction, protein contaminants are denatured and partition either with the organic phase or at the interface between organic and aqueous phases, while nucleic acids remain in the aqueous phase. Phenol used in this protocol is buffered to prevent oxidized products in the phenol from damaging the nucleic acids. Be aware that phenol can cause severe chemical burns on skin and will damage clothing. Wear gloves, safety glasses, and a laboratory coat when working with phenol. In the method presented here, phenol/chloroform (50%/50%; v/v) is recommended for extraction. In most cases, this mixture provide good protein denaturation and a tighter interphase between the aqueous and organic phases. If there is a problem with excessive foaming during the extraction, isoamyl alcohol can be added to obtain an organic composition of phenol/chloroform (50%/49%)/isoamyl alcohol (I%).

Conventional Ethanol Precipitation

During the ethanol precipitation, salts and other solutes such as residual phenol and chloroform remain in solution while nucleic acids form a white precipitate that can,easily be collected by centrifugation. If the aqueous volume is less than 450 pL, the reaction can be performed in a microcentrifuge tube. This is the most convenient format for performing organic extractions and ethanol precipitations. For larger volumes, multiple microcentrifuge tubes can be used, or the reaction can be scaled up. When scaling the reaction up, use tightly capped polypropylene tubes for the phenol/chloroform extraction, and centrifuge at 2500 rpm at room temperature to resolve phases. Polystyrene tubes cannot withstand the phenol/chloroform. Ethanol precipitation can be performed in 15- or 30-mL Corex tubes, and the precipitate collected by centrifugation at 10,000 x g for 15 min at 4 OC. It is recommended that Corex tubes be acid washed before use by immersion in 50% nitric acid for 1 hr, followed by thorough rinsing in distilled water, and autoclaving for 20 min.

In our hands, nucleic acid fragments and oligonucleotides longer than 15 nucleotides can be efficiently precipitated using this protocol. For efficient precipitation, the nucleic acid concentration should be at least 10 gg/mL. Lower concentrations of nucleic acids can be precipitated, but the recovery may not be quantitative. To precipitate lower nucleic acid concentrations, incubate the precipitate at -20 'C (or on dry ice) for 4 hr to overnight, and centrifuge for 30 min to collect the precipitate. Alternatively, nanogram quantities of nucleic acid can be efficiently precipitated by adding yeast tRNA carrier to the solution to obtain a nucleic acid concentration of 10 @g/mL before initiating the extraction and precipitation procedure. The presence of TRNA carrier is typically not a problem, except when the carrier will interfere with subsequent enzymatic manipulations of the sample (eg, if a DNA fragment will be end-labeled with T4 polynucleotide kinase).

The recommended salt for most routine applications of this method is 0.3 M sodium acetate (final concentration), which is more soluble in ethanol than 0.3 M sodium chloride and therefore less likely to precipitate with the nucleic acid sample. For samples containing sodium dodecyl sulfate (SDS), the recommended salt is 0.2 M sodium chloride, since the SDS is soluble in ethanol under these conditions. For removal of triphosphates (labeled or otherwise), 2 M ammonium acetate is recommended instead of 0.3 M sodium acetate, since triphosphates are less likely to precipitate under these conditions. Ammonium acetate is not recommended if the nucleic acid sample will be 5' phosphorylated by T4 kinase or tailed at the 3' end with terminal transferase, since residual ammonium ions will inhibit these two enzymes. Alternatively, LiCl can be used as the salt for precipitation. Instead of addition of 1/10 volume 3 M sodium acetate, add 1/10 volume 8 M LiCl. Since LiCl is very soluble in ethanol, the resulting precipitate is relatively salt-free. LiCl should be avoided, however, if precipitated RNA will be used as template for reverse transcription after precipitation.

Two Variations of the Precipitation Procedure

If it is desirable to keep the volume of the precipitating nucleic acids to a minimum, isopropanol at a volume equal to the volume of the aqueous DNA solution can be substituted for the ethanol in the precipitation reaction. With this substitution, precipitation can be performed from a starting aqueous volume of 700 liL in a single microcentrifuge tube. Isopropanol is not as volatile as ethanol, and is therefore more difficult to remove by evaporation in a vacuum centrifuge. Some salts are less soluble in isopropanol, and may be precipitated with the nucleic acids. It is recommended that isopropanol precipitation be followed immediately by a conventional ethanol precipitation to eliminate residual isopropanol and salt.

Isolation of DNA

Because of the large size and the fragile nature of chromosomal DNA, it is unlikely that anyone has ever isolated it in an intact, undamaged form. Several isolation procedures have been developed that provide DNA in a biologically active form, but this does not mean it is completely undamaged. These DNA preparations are stable, of high molecular weight and relatively free of RNA and protein. Here, a general method will be described for the isolation of DNA in a stable, biologically active form from microorganisms. The procedure outlined is applicable to many microorganisms and can be modified as necessary.

Designing an isolation procedure for DNA requires extensive knowledge of the chemical stability of DNA as well as its condition in the cellular environment. Several chemical bonds may be susceptible to cleavage during the extraction process. The experimental factors that must be considered and their effects on various structural aspects of intact DNA are outlined below.

1. pH

(a) Hydrogen bonding between the complementary strands is stable between pH 4 and 10.

(b) The phosphodiester linkages in the DNA backbone are stable between pH 3 and 12.

(c) N-glycoside bonds to purine bases (adenine and guanine) are hydrolyzed at pH values of 3 and less.

2. Temperature

(a) There is considerable variation in the temperature stability of the hydrogen bonds in the double helix, but most DNA will begin to unwind in the range of 80-90'C.

  1. Phosphodiester linkages and N-glycoside bonds are stable up to IOOOC.





3. Ionic Strength

(a) DNA is most stable and soluble in salt solutions. Salt concentrations of less than 0.1 M weaken the hydrogen bonding between complementary strands.

4. Cellular Conditions

(a) Before the DNA can be released, the bacterial cell wall must be lysed. The ease with which the cell wall is disrupted varies from organism to organism. In some cases (yeast), extensive grinding or sonic treatment is required, whereas in others (B. subtilis), enzyme hydrolysis of the cell wall is possible.

(b) Several enzymes are present in the cell that may act to degrade DNA, but the most serious damage is caused by the deoxyribonucleases. These enzymes catalyze the hydrolysis of phosphodiester linkages.

(c) Native DNA is present in the cell as DNA-protein complexes. The proteins (basic proteins called histones) must be dissociated during the extraction process.

5. Mechanical Stress on the DNA

(a) Gentle manipulations may not always be possible during the isolation process. Grinding, shaking, stirring, and other disruptive procedures may cause cleavage (shearing or scission) of the DNA chains. This usually does not cause damage to the secondary structure of the DNA, but it does reduce the length of the molecules.

Now that these factors are understood, a general procedure of DNA extraction may be outlined:

Step 1. Disruption of the cell membrane and release of the DNA into a medium in which it is soluble and protected from degradation

The isolation procedure described here calls for the use of an enzyme, lysozyme, to disrupt the cell membrane. Lysozyme catalyzes the hydrolysis of glycosidic bonds in cell wall carbohydrates, thus causing destruction of the outer membrane and release of DNA and other cellular components. The medium for solution of DNA is a buffered, saline solution containing EDTA. DNA, because it is ionic, is more soluble and stable in salt solution than in distilled water. The EDTA serves at least two purposes. First, it binds divalent metal ions (Cd", Mg 2 +, Mn 2 +) that could form salts with the anionic phosphate groups of the DNA. Second, it inhibits deoxyribonucleases that have a requirement for Mg2+ or Mn 2 1. Citrate has occasionally been used as a chelating agent for DNA extraction; however, it is not an effective agent for binding Mn 2 '. The mildly alkaline medium (pH 8) acts to reduce electrostatic interaction between DNA and the basic histones and the polycationic amines, spermine and spermidine (see Experiment 21). The relatively high pH also tends to diminish nuclease activity and denature other proteins.

Step 2. Dissociation of the protein-DNA complexes

Detergents are used at this stage to disrupt the ionic interactions between positively charged histones and the negatively charged backbone of DNA. Sodium dodecyl sulfate (SDS), an anionic detergent, binds to proteins and gives them extensive anionic character. A secondary action of SDS is to act as a denaturant of deoxyribonucleases and other proteins. Also favoring dissociation of protein-DNA complexes is the alkaline pH, which reduces the positive character of the histones. To ensure complete dissociation of the DNA-protein complex and to remove bound cationic amines, a high concentration of a salt (NaCl or sodium perchlorate) is added. The salt acts by diminishing the ionic interactions between DNA and cations.








Step 3. Separation of the DNA from other soluble cellular components

Before DNA is precipitated, the solution must be deproteinized. This is brought about by treatment with chloroform/isoamyl alcohol and followed by centrifugation. Upon centrifugation, three layers are produced:

an upper aqueous phase, a lower organic layer, and a compact band of denatured protein at the interface between the aqueous and organic phases. Chloroform causes surface denaturation of proteins. Isoamyl alcohol reduces foaming and stabilizes the interface between the aqueous phase and the organic phases where the protein collects.

The upper aqueous phase containing nucleic acids is then separated and the DNA precipitated by addition of ethanol. Because of the ionic nature of DNA, it becomes insoluble if the aqeuous medium is made less polar by addition of an organic solvent. The DNA forms a threadlike precipitate that can be collected by "spooling" onto a glass rod. The isolated DNA may still be contaminated with protein and RNA. Protein can be removed by dissolving the spooled DNA in saline medium and repeating the chloroform/isoamyl alcohol treatment until no more denatured protein collects at the interface.

RNA does not normally precipitate like DNA, but it could still be a minor contaminant. RNA may be degraded during the procedure by treatment with ribonuclease after the first or second deproteinization steps. Alternatively, DNA may be precipitated with isopropanol, which leaves RNA in solution. Removal of RNA sometimes makes it possible to denature more protein using chloroform/isoamyl alcohol. If DNA in a highly purified state is required, several deproteinization and alcohol precipitation steps may be carried out. It is estimated that up to 50% of the cellular DNA is isolated . The average yield is 1 to 2 mg per gram of wet packed cells.


Characterization of DNA

DNA has significant absorption in the UV range because of the presence of the aromatic bases, adenine, guanine, cytosine, and thymine. This provides a useful probe into DNA structure because structural changes such as helix unwinding affect the extent of absorption. In addition, absorption measurements are used as an indication of DNA purity. The major absorption band for purified DNA peaks at about 260 nm. Protein material, the primary contaminant in DNA, has a peak absorption at 280 nm. The ratio A260/A280 is often used as a relative measure of the nucleic acid/protein content of a DNA sample. The typical A260/A280 for isolated DNA is 1.9. A smaller ratio indicates increased contamination by protein.