Nucleic acid extraction methods are key to molecular biology, and are routinely utilized in many applications in both medical and biological sciences. Nucleic acids were first extracted in 1869 by Friedrich Miescher, when he was studying the chemical nature of the nuclei of white blood cells. In 1953, Rosalind Franklin, James Watson, and Francis Crick determined the structure of deoxyribonucleic acid (DNA), winning James Watson, Francis Crick, and Maurice Wilkins (Rosalind Franklin’s boss) the Nobel Prize in Medicine in 1962. Their work showed that DNA was made up of two strands of long chains of individual nucleic acid molecules (called nucleotides) wound around each other in a helix. This discovery made it very clear that DNA was a perfect molecule for storing, and copying vast amounts of information – making it ideally suited to store the “blueprints” for cells, and therefore life forms. Later, in 1968, Gobind Khorana, along with Marshall W. Nirenberg and Robert W. Holley, won the Nobel Prize for demonstrating how the order (sequence) in which the nucleotides are joined together in strands of ribonucleic acid (RNA) could be translated into chains of amino acids to make proteins – one of the primary building blocks of cells. Collectively these discoveries, as well as many others, have given us a clear understanding the kinds of information that are stored in cells, how this information is copied, and how it is used to direct the formation and metabolisms of living things. Further studies of nucleic acid molecules form the core of the field of molecular biology, which has allowed us to discover disease-related DNA sequences, including those related to cancer, genetic diseases, and microbial infection. Molecular biology has also shed light on virtually every aspect of the life sciences.
Nucleic acid extraction can be divided into 3 steps, which can be optimized depending on the sample type and downstream applications for which the nucleic acids will be used. The steps are:
1) Breaking open the tissues and cells
2) Removing proteins, lipids and other contaminants from the nucleic acids
3) Transferring the nucleic acids to water, or a buffer solution that will preserve them without interfering with subsequent work
Nucleic acid extraction methods
Nucleic extraction methods can be widely characterized into two different types:
- Solution-based methods
- Solid-phase based methods
Many of the early methods for nucleic acid extraction depended on grinding frozen biological samples, and then mixing them with solutions of chemicals devised to make it possible to purify RNA and/or DNA.
1. Cesium Chloride Gradient Centrifugation (with Ethidium Bromide)
This method is based on the phenomena of buoyant and specific density. Cesium chloride (CsCl) is an extremely dense salt. When solutions of this salt are subjected to centrifugation at very high speeds (typically >100,000 rpm), the CsCl sets up a concentration gradient, where it is highly concentrated at one end, or side, of the centrifuge tube that it’s in, and much less concentrated at the other. If the centrifugation process is ended gently, with the centrifuge slowing down slowly, the denser solution (with a higher CsCl concentration) will settle to the bottom of the tube, maintaining the concentration gradient for some time. Other molecules dissolved in the solution will separate themselves according to how dense they are, with the denser molecules going toward the bottom of the tube, and less dense molecules moving toward the top. When DNA is in the presence of a molecule called ethidium bromide, it will bind to it and become fluorescent when under UV light. The ethidium bromide also adjusts the density of DNA so that it moves toward the center of the tube upon centrifugation. Contaminants will move to different positions, and therefore will be separated from the DNA, which can be easily seen and recovered since it will be fluorescent. Subsequent steps will then separate the DNA from the CsCl and ethidium bromide. This technique is particularly effective for separating plasmids – which are small DNA strands that bacteria use to share information on antibiotic resistance with each other, and mitochondrial DNA, and other specific DNA molecules that bind to characteristic amounts of ethidium bromide, and therefore will have unique densities. It is easy to separate mitochondrial and plasmid DNA. These nucleic acids are arranged in highly coiled (called supercoiled) circular patterns. DNA that is not supercoiled (like much longer chromosomal DNA species) bind to different amounts of ethidium bromide, and have a different density – making them easy to separate from the plasmid DNA.
2. Guanidinium Thiocyanate-Phenol-Chloroform nucleic acid extraction
An aqueous solution of the salt guanidinium thiocyanate, when mixed with solvents phenol and chloroform, allows for effective purification of RNA through a minimal number of steps. When samples are ground up and mixed with chloroform, phenol, sodium acetate and guanidinium thiocyanate, and subjected to centrifugation, the salt solution will separate from the solvents – providing an upper aqueous phase and a lower organic phase that consists of the solvents. RNA will remain in the aqueous phase, while proteins, other nucleic acids, and other contaminants will move to the organic phase or to the interface between the two phases.
3. Cetyltrimethylammonium Bromide nucleic acid extraction
This technique is mostly used for plant samples as well as their parts like grains, seeds and leaves. It is also used for many food samples. Nucleic acids, and some other polysaccharides are insoluble in solutions of 2% Cetyltrimethylammonium Bromide nucleic acid extraction (CTAB), at high pH. Neutral polysaccharides and proteins are soluble. This provides an easy way to remove many difficult plant-based contaminants prior to further purification.
4. Chelex® Extraction
This technique is utilized in the field of forensics for DNA extraction from different sources like buccal swabs, blood stain cards and hair. This method uses a resin (trade named Chelex®, that binds to common inhibitors of the polymerase chain reaction (PCR) process. This yields a fairly crude sample, but one that preserves DNA and renders it useful for PCR-based forensic analysis.
5. Alkaline Extraction
This technique is used for plasmid DNA isolation, by itself for relatively crude preparations, or as the first step in virtually all plasmid purification processes. It involves harvesting bacteria of interest from a culture media and consequently exposing the bacteria to a highly alkaline solution. This is generally followed by mixing the alkaline extract with the detergent Sodium Dodecyl Sulfate, which removes proteins and most other contaminants. One of the biggest advantages of this methods is that, since there are many proteins bound to the large chromosomal DNA, this DNA is removed along with the proteins. Since molecular cloning techniques depend on manipulating plasmid DNA, removing the chromosomal DNA is critical.
Solid-phase nucleic acid extraction
Solid phase extraction methods work by causing nucleic acids to bind to solid supports, such as magnetic beads coated with silica or other materials. The beads (or other supports) are then washed with alcohol to remove contaminants. The support is then washed with a liquid that renders the nucleic acids soluble again, which frees the DNA from the support in a process known as elution. A simple method is to add a solution containing a chaotrope to the crude nucleic acid extract. Chaotropic agents are molecules that disrupt hydrogen bonding network between water molecules. In the presence of chaotropes, nucleic acids before far less soluble and will bind to glass, silica-coated magnetic beads and other solid supports – making it easy to separate them from contaminants. Solid phase extraction methods can also depend on selective binding of DNA and RNA to ion-exchange resins or other chemistries.
There are numerous devices that are used in nucleic acid extraction methods, these are Spin Columns, Beads or Magnetic Beads, Automated Nucleic Acid Extraction Systems or Liquid Handling Robots as well as Lab-on-a-Chip Cartridges and Microfluidics.
Nucleic acid extraction is an important part of molecular biology. There have been several modifications to the original method which was developed in 1869. Both the mechanical and chemical processes have peculiarities that affect their use, particularly point-of-care diagnostics.