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Comparing Nucleic Acid Purification Methods: Phenol-Chloroform, Silica Spin Column, Magnetic Beads

Updated: Mar 11

There are several techniques that can be used to extract and purify nucleic acids from biological samples to be used for downstream applications. The three most common nucleic acid purification techniques are phenol-chloroform extraction, silica spin column purification, and silica coated magnetic bead extraction. A number of factors need to be considered to determine which technique is best suited for your specific application: maximum product yield, product purity, speed of the procedure, and cost effectiveness.

Phenol-chloroform Extraction

Fig 1. Trizol Separation Phase

Phenol-chloroform phase separation is the traditional method used to purify nucleic acids from biological samples. Trizol, which is specifically designed to purify RNA molecules, is the best known acid-guanidinium phenol reagent used in phenol-chloroform extraction. In the purification procedure the biological sample is first mixed with the Trizol reagent. Trizol lysis the tissues and cells and releases nucleic acids and proteins into the solution. The mixture is then mixed with the liquid organic chloroform and allowed to settle. This forms 2 distinct aqueous and organic phases together with the interphase layer (Fig. 1). The aqueous phase contains RNA, organic phase - proteins, and DNA molecules settle at the interphase. The aqueous phase is then carefully removed with a pipette tip to collect the extracted RNA. Finally, RNA is washed to remove salts and concentrated by a few rounds of ethanol precipitation (Fig. 2)

Fig 2. Ethanol precipitation

The main components of Trizol are guanidinium salts, phenol, staining dye, and pH-controlling buffer. Guanidinium salts are chaotropic agents that lyse the tissue and cells to release the nucleic acids and proteins into the solution. They also acts to denature the proteins present in the sample. This exposes hydrophobic parts of the protein, which causes them to partition into the organic phase. The phenol component of Trizol facilitate s the separation of and better defined interface formation between the two phases, and forms part of the organic phase. The pH buffer component of Trizol is used to keep pH of the solution at ~4. At this pH RNA molecules remain highly charged, while the charge of the DNA molecules in partially neutralized. As a result RNA remains in the aqueous phase, while the less charged DNA is partitioned into the interphase layer. Lastly, the red hydrophobic dye stains the organic phase red, making it easy to visualize.

A major benefit of using Trizol and other phenol-chloroform purification techniques is their low cost. Furthermore, when done properly, this technique can have essentially no loss of nucleic acids. Both long and short nucleic acids can be retrieved with similar efficiency. However, a major limitation is that the need to wait for the phase separation, careful phase removal, and the need for multiple ethanol precipitation steps makes this procedure quite long. Unless the separation is carried our very precisely, intermixing and cross contamination between the aqueous, organic and interphase layers can happen. Furthermore, phenol and chloroform are toxic compounds and care needs to be taken during the procedure to avoid accidental exposure of skin or inhalation. Lastly, this technique cannot be easily adapted to high through-put applications due to the requirement of complex, mechanical pipetting during phase separation.

Silica Spin Columns

Fig. 3: Nucleic acid purification with silica spin columns.

An alternative technology uses silica spin columns to extract and purify nucleic acids from biological sample. The purification starts by resuspending the biological sample in the lysis buffer (Fig. 3, Step 1). Lysis buffer contains high concentration of guanidinium salts, which lyse the tissues and cells, releasing nucleic acids and proteins in the solution. In Step 2, ethanol or isopropanol is added to the lysed solution, and the mixture is flown through the silica column, either using centrifugal force in a centrifuge, or by application of vacuum. Silica columns have a membrane that is made up of multiple layers of silica. Guanidinium salts also act to form the bridges between the negatively-charged nucleic acid backbone and the silica membrane. The addition of ethanol or isopropanol increases the hydrophobicity of the buffer and drives the nucleic acid to interact with the silica membrane. This results in a stable loading of nucleic acids onto the column, while proteins and other cellular components pass through the column. Conditions of the buffers (such as specific salts and their concentration used, as well as pH) can be adjusted to preferentially bind DNA or RNA to the column.

Once the nucleic acid has been bound to the membrane, it is washed with ethanol containing wash buffer to remove any residual contaminants (proteins, salts, cell residues) that may be present. Finally, elution buffer or ultra-pure water is added to the column to elute the purified nucleic acid.

Silica Spin Columns offer a number of advantages over phenol-chloroform extraction. Purification of nucleic acids with silica spin columns is much faster and can be completed in as little as 15 minutes after prepping the columns with the solution. The procedure is much simpler and less prone to experimental error, so cross-contamination of the sample is much less common. Therefore, the extracted nucleic acid product is usually of higher purity. Silica spin column purification does not use dangerous phenol and chloroform chemicals, making this method much safer. The limitation of silica spin column technology is that it makes the purification more costly when comparing to using cheaper phenol-chloroform and Trizol chemicals. There is generally some loss of nucleic acids during the column purification. This is particularly true for shorter nucleic acids, such as siRNA (though buffer formulations and silica membrane modifications have been developed to enhance the binding of smaller molecules to the column).Silica column format is also not particularly suitable for high throughput automation. However, with devices such as Qiagen's QIAcube, extraction using multiple columns in parallel can be automated. Alternatively, silica spin columns are also available in 96-well plate format, where each well of a 96-well plate contains an individual silica spin column. With 96-well plate format 96 samples can be extracted in parallel using a centrifuge or a vacuum manifold. Extraction in the 96-well plate format can be further automated by combining it with a positive pressure liquid handling device such as Tecan's Resolvex system.

In general, a lab that has a small sample size would find it convenient to use the simple and safe silica spin column purification as it will yield a high amount of pure nucleic acid with lower chance of contamination compared to the phenol-chloroform method. For a lab that needs to extract a larger number of samples, a 96-well silica spin column plate format might be be preferable.

Luna Nanotech offers a range of Nucleic Acid Purification Kits both in single silica spin column as well as 96-well plate Silica Spin Column formats. Browse our products here, or take a look at our catalog.

Silica Coated Magnetic Beads

The last method for the extraction and purification of nucleic acids uses silica coated magnetic beads. The beads consist of a paramagnetic core surrounded by an outer layer of silica. The paramagnetic nature of the beads means that they are not attracted to each other but are all drawn to an external magnetic field. The silica coat makes the beads similar to Silica Spin Columns in their ability to bind nucleic acids. The main difference being that the silica is conjugated to the surface of the beads rather than being anchored in the column on a layered silica membrane. The buffers and purification steps are similar to those used with the silica spin columns (Fig. 4). A lysis buffer is first added to lyse the sample (Fig. 4, Step 1). Silica coated magnetic beads are then mixed in together with the ethanol or isopropanol (Step 2). The nucleic acids are loaded onto the magnetic beads through the chaotropic salts induced formation of the salt bridges between the nucleic acid backbone and magnetic beads' silica coat. The sample is then placed onto the magnetic rack to immobilize the beads on the wall of the tube, allowing the supernatant to be removed (Step 3, magnetic separation). The wash buffer is then added to remove any remaining protein and salt contaminants (Step 4), followed by another round of magnetic separation to discard the used washed buffer supernatant (Step 5). Following the wash steps, the elution buffer is added to the beads to released purified nucleic acid into solution (Step 6). The final round of magnetic separation is used to immobilize the beads and remove the supernatant containing the purified nucleic acid (Step 7).

Fig 4. Nucleic Acid Purification with silica-coated magnetic beads.

The yield, purity and cost of silica coated magnetic bead technique is similar to that of silica spin columns. However, the main advantages of using magnetic beads for nucleic acid purification are speed and simplicity. The purification can be performed on bench top and does not require any equipment such as a centrifuge or a vacuum manifold. Each magnetic separation step can be as short as 30 seconds, making this the fastest nucleic acid purification technique. For example, complete PCR product purification can be completed in under 5 minutes!

Magnetic bead purification is also particularly suitable for high throughput applications and automation. Magnetic bead purification can be performed in a 96-well deep well plate format, allowing 96 samples to be co-purified in parallel. Since magnetic separation creates two very clearly distinct phases: solid magnetic beads held at the surface by a magnetic field and a liquid supernatant, liquid handling robotic systems can be easily designed for automated bead manipulation and supernatant removal. In fact, high throughput magnetic purification of viral RNA with such automated systems as Hamilton STAR, ThermoFisher KingFisher, and others was the most common method used in the diagnostic of SARS-CoV-2 during the pandemic.

Luna Nanotech offers a full line of silica coated magnetic bead based kits in a number of different formats. You can browse our products here, or take a look at our catalog. Luna Nanotech also provides a full range of magnetic racks.

Are you tired of filling your own 96-well plates with the purification reagents? Ask us about our kits that come with 96-well plates already prefilled with all of the reagents!

Are you looking for the fastest way to purify plasmid DNA on MAXI scale? Check out our PuroMAG™ MAXI Plasmid Purification Kit. 2 minute magnetic separation steps and no equipment required!

If you are looking for more information or need help deciding which technique is best for your lab, please do not hesitate to contact us, we are always happy to help!

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