Nucleic Acid Extraction: Overview of Most Used Methods

Nucleic Acid Extraction: Overview of Most Used Methods

Nucleic acid extraction, which entails both nucleic acid isolation and nucleic acid purification, is a critical step for many molecular biology applications, such as PCR, sequencing, molecular cloning, and diagnostic testing. Obtaining high yields of high quality extracted nucleic acid can help ensure that your downstream experiments will be successful.

In this article, we provide an overview of the most commonly used methods for DNA and RNA extraction, as well as some of the advantages and disadvantages of each method.

Deciding which extraction method is best for you depends on several factors, such as your downstream applications and the purity of DNA or RNA that is needed, the volume of your sample, as well as your available lab equipment, budget, and time.

All Extraction Techniques Share Common Steps

There are several methods for extracting pure DNA or RNA and they all share some basic steps:

Cell lysis whereby lipid membranes are disrupted by chemical (eg, detergents, proteases) and/or mechanical means to allow the release of nucleic acids
Inactivation of nucleases to ensure that the extracted nucleic acid is not degraded
Purification to separate the nucleic acid from cellular debris

Extraction Methods Can be Divided into Solution-based or Solid-phase Methods

1. Solution-Based Extraction Method: Phenol-Chloroform Extraction

With this method, after cells are lysed and nucleases are inactivated, a phenol-chloroform-isoamyl alcohol solution is added to the lysed cells, mixed well, and then centrifuged. After centrifugation, 3 layers will be seen: an upper aqueous phase containing DNA (due to the hydrophilic nature of DNA), a lower organic phase of lipids, and an interphase of denatured proteins. The aqueous phase is collected and a high salt buffer with ethanol or isopropanol is used to precipitate the DNA. The DNA is collected by centrifugation and then washed and resuspended in buffer or water for storage.

If the aim is to extract RNA, the pH of the solution must be slightly acidic. At pH 4 – 6.5, DNA will be retained in the lower organic phase and RNA will remain stay in the upper aqueous layer. RNA can also be separated from DNA after extraction using an acidic solution of guanidinium thiocyanate, sodium acetate, phenol and chloroform. The RNA is then collected by precipitation with ethanol or isopropanol.

Advantages of the phenol-chloroform extraction method include the capacity to obtain high yields, and the efficient extraction of both long and short nucleic acids. This is the most effective method for obtaining high molecular weight DNA. Other advantages include that there are no requirements for specialized lab equipment, and the low cost of the procedure. The disadvantages associated with this extraction method are due to it being performed manually, which can lead to higher variability and lower reproducibility. This method is also more time consuming compared with other extraction methods, cannot be easily scaled up, and requires the use of toxic reagents.

Solid-phase Extraction Methods: Spin Column Extraction and Magnetic Bead Extraction

In these techniques, DNA or RNA selectively binds to a solid matrix (ie, a column or beads) under appropriate buffer conditions. The matrix is then washed to remove unwanted cellular components (contaminants) before the nucleic acid is detached from the solid matrix with an elution buffer and is collected. Silica (glass fibers) is usually used as the binding matrix for DNA extractions. DNA and silica are both negatively charged, thus normally they repel each other and bind to water via a hydrogen bond. When buffers containing chaotropic salt and ethanol are present, the DNA-water bond is weakened, causing the DNA to bind to the silica. Alcohol in the buffer increases the hydrophobicity, further stabilizing DNA to the silica. The DNA can later be eluted off the silica under low salt conditions.

Altering buffer conditions (eg, salt concentration/pH level) and substituting other binding substrates for the silica can allow for the selective binding of RNA rather than DNA. It is also possible to extract both DNA and RNA together under appropriate conditions. The substrate and buffer conditions will depend on the nucleic acids you wish to extract for downstream applications.

Spin Column Extraction

Spin column extractions are usually performed with a specific kit, many of which are commercially available. Choosing the most effective spin column kit will depend upon your sample and your intended downstream applications. This technique uses a solid matrix (eg, silica) that is packed into a column. The matrix will selectively bind to your desired nucleic acids.

For DNA extractions, after the cells are lysed, the cell lysate, in buffer containing chaotropic salt and ethanol, is transferred to a spin column and centrifuged or subjected to a vacuum. The DNA in the sample will bind to the silica membrane inside the column while other cellular components pass through the column. The column is then treated with wash buffer and centrifugation several times to release any contaminates from the column. An elution buffer (low salt buffer or water) is then added to the column. The buffer rehydrates the DNA and frees it from the membrane so it can be collected in the base of the spin column after centrifugation.

Prior to adding the cell lysate, the spin column is conditioned for the appropriate nucleic acid absorption with buffer of an ideal pH. The desired nucleic acids absorb to the column because of the pH of the binding solution.

Advantages of spin column extraction include its ease and speed to perform. Because of its simplicity, this method is less prone to errors in manipulation and the extracted nucleic acid is usually of high purity. The protocol can be used for a few or many samples: Spin columns are available as individual columns, if you have only a few samples, or as 96-well plates, which can be placed on a vacuum manifold in place of centrifuging, if you have many samples. Disadvantages include the possibility of membrane clogging, and a required minimum elution volume of about 50 ml, which can result in a lower DNA concentration. Additionally, there is often some loss of nucleic acid, especially for shorter nucleic acids.

Magnetic Bead Extraction

This newer method of extraction is also performed with specific commercially available kits, which allow extraction of DNA, RNA or total nucleic acid. This extraction technique utilizes tiny magnetic beads comprised of a paramagnetic core surrounded by a layer of binding matrix (such as silica) that can reversibly bind nucleic acids under certain buffer conditions. The beads are not attracted to one another, and therefore can be separated in suspension; they are only attracted to an external magnetic field. After the sample is lysed, it is incubated with the silica (or other substrate)-coated magnetic beads in the appropriate binding buffer. The nucleic acids will bind the silica surface of the beads, in a manner similar to how they bind the silica in spin columns. The buffers and purification steps are also similar to those used with silica spin columns. The tubes containing sample and beads are placed on a strong magnet to attract and hold the beads (which have bound nucleic acids) in place on the side of the tubes while the supernantant containing cellular debris is aspired. One or more cycles of washing the beads and aspirating the supernantant are performed to further remove contaminants. An elution buffer is then added to release the nucleic acids from the beads and the beads are held in place with an external magnet once again, so that the purified nucleic acid can be collected.

Advantages of magnetic bead extractions include its speed (it is the quickest nucleic acid extraction technique) and its simplicity, as well as the capability to elute nucleic acid with small volumes. The protocol can also be adapted for large numbers of samples, with the use of 96- or 384-well formats. Magnetic bead extraction is suitable for high throughput applications and automation. Disadvantages of the procedure when done manually include the possibility of aspirating the beads and contaminating the isolated nucleic acid. Yield may also be less than that of other purification methods.

Automated Extraction Instruments or Outsourcing Can Have Advantages Over Manual Extractions

Automated and semi-automated nucleic acid extraction instruments offer several advantages over manual techniques including scalability, reliability and reproducibility, and less hands-on time. Outsourcing your nucleic acid extractions is another option that can save you valuable lab time and can provide the advantages of automation while avoiding the upfront cost of purchasing automated instruments.

Conclusion

Various nucleic acid extraction methods are available. When choosing the method that will serve you best, you must consider how you will be using the nucleic acids and how purified the samples need to be. Additionally, you must consider the number of samples that require extraction, and the capabilities of your lab – do you have the necessary instrumentation and experienced personnel? Do you have the time? The right extraction kits and automated instruments can help ensure successful and efficient extractions for your downstream applications.

If your lab is short on instruments, personnel, or time, you may find that outsourcing your nucleic acid extractions is the answer. An outsourced extraction specialist can provide expert nucleic acid extractions for all your downstream applications.

Have questions about your DNA or RNA extraction projects? Talk to AutoGen. We’re always happy to help you make the best decisions for your individual needs.