DNA extraction has been extensively studied and many different methods have been developed. To extract DNA from samples, such as blood, cells, microbes, soil, plants or animal tissues, the samples must go through various manipulations which depend on the sample and downstream application. These include cell lysis, inactivation of nucleases, and purification.
Isolated DNA from samples can be used for a variety of downstream applications. It is extremely important to ensure the quality and quantity of the isolated DNA to successfully complete the downstream applications. Time, cost and expertise should also be considered when choosing your extraction method. To extract a sufficient yield of high quality, purified DNA for your downstream applications, you have to find the best extraction method for your sample type and lab. Each sample type needs a different DNA extraction setup, which can make things difficult. In this article, we will discuss DNA extraction methods, comparing the methods for a better understanding.
Chemical DNA extraction methods
Organic DNA extraction
Phenol-chloroform and isoamyl alcohol (PCI)
First, cells are treated with a lysis buffer containing salts to dissolve cell membranes and the nuclear envelope. Then, the lysed samples are mixed with a Phenol-chloroform solution. The solution is then centrifuged for a few minutes. The emulsion separates into three phases during centrifugation - the upper aqueous phase, the bottom organic phase of lipids and an interphase comprising proteins. The aqueous portion is transferred to a new tube with a pipette, and the organic phase can be discarded, ethanol precipitation is then used to purify and concentrate the DNA.
This method is considered one of the best for DNA extraction, as it can be used to extract DNA from blood, suspension culture, and nearly all tissue types. It also provides high quality, high yields and is fast, but the highly toxic agents, such as phenol and chloroform, are harmful which necessitate the use of fume hoods and create a major limitation to this method. It requires training to prepare and handle these chemicals. It is also time consuming and needs to be performed manually, leading to higher variability and lower reproducibility.
Inorganic DNA extraction
Proteinase K DNA extraction
Proteinase K is one of the most common protease enzymes used in DNA extraction. 20–50 μL of 10–20 mg/mL proteinase K is added for DNA extraction, then sodium dodecyl sulfate (SDS) is also added to dissolve the cell membrane and nuclear envelope in addition to denaturing and unfolding proteins, which exposes them to the protease activity of proteinase K. The solution is incubated then can be used to extract DNA using the salting-out method or phenolchloroform.
This method is easy to use and is safe and accurate. It also provides high quality and a high quantity of DNA for a wide range of samples; however it takes a long time and can be costly. Also, if the enzyme proteinase is not maintained well in a cold chain it cannot be utilized for a long period of time and the stability of the enzyme is low as well.
CTAB DNA extraction
Cetyl Trimethyl Ammonium Bromide (CTAB) is a chemical used for DNA extraction. The samples are added to 2% CTAB at alkaline PH. In a low ionic strength solution the extraction buffer precipitates DNA and acidic polysaccharides from other cellular components. Then, high salt concentration solutions are used to remove DNA from the acidic polysaccharides which form a precipitate with CTAB.
This method is especially useful for DNA extraction from plants and bacteria that produce high amounts of polysaccharides. The DNA is purified using a variety of organic solvents and alcohols, this includes toxic agents - for example phenol and chloroform. This method provides high quality and high yields, but the downside to this method is the use of toxic reagents as well as its time-consuming protocol and extensive chemical preparation. It also requires additional techniques like tissue homogenization and the use of liquid nitrogen.
SDS DNA extraction
SDS is an anionic detergent that digests nuclear and cell membrane proteins.
SDS gives a negative charge to each protein depending on their size. Since all proteins have the same shape during gel separation, they are separated only due to their size. Therefore, SDS can be used to aid in lysing cells during DNA extraction.
Salting out method
A simple and non-toxic method, where the sample is added to the lysis buffer, SDS and proteinase K. Then the mix is incubated overnight, and then saturated NaCl is added, and the mixture is shaken then centrifuged. The high salt concentration decreases protein solubility, which results in precipitation. The supernatant is then pipetted into a new tube and can be precipitated using ethanol.
This method tends to yield high quality DNA. It is used to extract DNA from blood, suspension culture or tissue homogenate. It is safer than some other methods, but the quality might not be as good.
Silica-gel-based techniques
Lysis buffer, SDS and Proteinase K are added to the sample, then after incubation, this mixture is added to a tube with silica gel. Phenol-chloroform is then added at a ratio of 1:1 and the mixture is then shaken and centrifuged. This process traps the proteins and organic phase beneath the silica column, while allowing the aqueous phase, which contains the DNA, to stay above the layer of gel polymer. The aqueous layer is then decanted or pipetted to a new tube and dissolved in TE buffer.
By using silica gel, it increases the purity of the extracted DNA and helps prevent physical contact with the toxic reagents. DNA yield is also higher than organic solvent-based DNA extraction methods. However, it can be costly.
Physical DNA extraction methods
Magnetic bead DNA extraction
For this method you need a lysis buffer and, depending on the sample, a physical lysis method to disrupt the cells. Then, magnetic beads that bind to the nucleic acids of the samples are added. Magnetic nanoparticles coated with a DNA binding antibody or polymer can be used to bind DNA to its surface. Magnetic beads are usually made of magnetite or maghemite at the core, and substances including silica as well as functional groups such as sulphate and hydroxyl groups. By applying a magnetic field at the bottom of the tube using an external magnet, separation of the DNA-bound magnetic beads from the cell lysate can be accomplished. With the beads grouped at the bottom of the tube, the supernatant can then be rinsed away. This step is repeated several times, replacing the wash buffer in between. Finally, the elution buffer is added to detach the nucleic acids from the beads, then the samples are transferred to a different vessel.
The DNA yield and quality of this method are good and the protocol is very fast. It is a very accurate technique and can handle a higher sample load. It is also safe and ideal for automation, plus it does not depend on centrifugation. Time and effort can be reduced exponentially with a dedicated system. Plus, steps are reduced and throughput increases. This method does not involve the use of shear forces that could damage the integrity of nucleic acids. However, manually performing this method is more tedious and error prone, as you have to be careful not to aspirate the magnetic beads. Therefore, this method is best used when automated.
Paper DNA extraction
Cellulose-based paper contains buffers, detergents, and chelating agents that enable DNA extraction. About 1-2 mm of the sample area is removed using a sterilized micropunch. Before being processed for downstream applications, the punch is washed with detergent and rinsed. Filter paper can also be used to isolate DNA from plant sources.
This type of extraction is fast, convenient, safe, cost effective, does not require much laboratory expertise and allows for easy storage of the sample. Yet, downstream processing to recover pure and concentrated DNA can be a challenge.
Scientists have made remarkable progress in designing extraction methods that are more reliable, easier, faster, more cost-effective and produce a higher yield. Newer techniques that are more reliable and efficient have helped advance knowledge in various fields in science. Most labs now extract DNA using sample and application specific kits for spin column or magnetic bead extraction.