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DNA is the unit of hereditary material in almost all organisms including humans. Mostly, each cell in a person’s body has the same content of DNA. Most of the DNA is located in the nucleus of the cell, which is called nuclear DNA. A smaller amount of DNA is present inside the mitochondrion, which is called mt DNA or mitochondrial DNA.
Initially, most of the time, DNA extraction was reliant on density gradient centrifugation. Until recently, most of the methods in the extraction of DNA were labor-intensive, complex, and time-consuming. Also, the methods provided smaller quantities of DNA. Nowadays, specialized extraction methods have been used with many of the methods being either column-based or solution-based. With the emergence of automation of the process and commercial kits, the extraction of DNA has become simplified. These changes have increased the production and yield of DNA.
As DNA is essential in the study of genetic causes of disease, DNA paternity tests, and for the development of drugs and diagnostics, the extraction of DNA is of utmost importance. Further, DNA is also a necessity in the detection of viruses and bacteria, DNA sequencing, and forensic science.
For the extraction of DNA, there are various steps and methods involved. This article brings out the steps and methods, which are implicated in the extraction of DNA.
For any extraction method, there are three basic steps for the extraction of DNA. They are lysis, Precipitation, and Purification.
The nucleus and cell are broken first for the release of DNA. Initially, mechanical disruption breaks open the cells. This is done by cutting the tissue into small pieces. In addition, small blender, mortar and pestle, and tissue homogenizer can be used too. Plant cells possess tough cell walls, so mechanical disruption is significant. For softer cell walls, detergents and enzymes like proteinase K are used to free the cellular proteins and DNA.
After lysis, DNA has been freed from the nucleus but mixed with cell parts. The precipitation step helps in the separation of DNA from the cellular debris. The negative charges present on the DNA molecules are neutralized by sodium ions, which render them more soluble. In the meantime, their solubility in water decreases. Then, alcohol like isopropanol or ethanol is added, causing the DNA to precipitate out of the aqueous solution as it is insoluble in alcohol.
Now, the DNA has been separated from the aqueous phase due to precipitation. It is rinsed with alcohol for the removal of cellular debris and remaining unwanted materials. The purified DNA is redissolved in water for storage and handling with ease.
In detail, there are five steps needed in extracting DNA in pure form. They are: the creation of lysate, clearing of the lysate, binding to the purification matrix, washing, and elution
Physical method, chemical method, enzymatic method, and combination of three methods are used for creating lysate.
The physical method is used for creating lysate from tough tissues. Freezing of cells and grinding with mortar and pestle under liquid nitrogen is performed and then, they are exposed to enzymatic or chemical lysis conditions. Grinders can be manual devices or advanced automation systems to disrupt multiple 96-well plates. It is used for plant tissues. Sonication can be used in lysis. Metallic or ceramic beads are also used for creating lysate.
The chemical method applies to easy-to-lyse materials like tissue-culture cells. A variety of agents are used for disruption of cell membranes and disruption of proteins. Detergents like SDS and chaotropes like alkaline solutions and guanidine salts are the chemicals generally used.
The enzymatic method is used along with other methods for disruption of plant materials, tissues, bacteria, and yeast. The enzymes disrupt tissues and tough cell walls. Lysozyme, liticase, zymolyase, collagenase, lipase, and proteinase K are the enzymes commonly used depending on the starting material.
Unwanted materials like lipids, proteins, and saccharides from cellular structures in the lysate can clog with membranes or interfere with downstream applications during the purification reaction. This clearing is established through filtration, centrifugation, or bead-based methods.
Centrifugation is suitable for clearing huge amounts of debris, but the process takes more time. Filtration is a speedy method, yet large amounts of debris can clog the membrane. Though bead-based clearing can be used in automated protocols, they are overwhelmed with biomass. After clearing is done, DNA is purified through different chemistries.
Silica, cellulose, and ion exchange matrices can be used for binding to genomic DNA, through which purification is performed. Each matrix has a characteristic binding capacity and the chemistry can influence the purity and efficiency of the DNA isolation. Binding capacity indicates how much nucleic acid can bind before the full capacity of the system is reached, so that further nucleic acid is not able to bind. The matrices can selectively bind to DNA, leaving out RNA and proteins.
The method is based on the interaction between positively charged particles and negatively charged particles that are present in the DNA. Under low salt conditions, DNA binds, and at high salt conditions, RNA and contaminating proteins can be washed out. DNA is eluted under high salt conditions and recovered through precipitation by ethanol.
In a cellulose-based matrix, nucleic acid binds to cellulose in the presence of alcohols and high salt. As the binding capacity is high, conditions are adjusted to bind different species and sizes of DNA preferentially. Due to relatively small elution volume, high concentration eluates for nucleic acids are obtained.
The high-concentration salt solution causes the protein to fall out of solution and centrifugation separates the soluble nucleic acid from precipitated protein and cell debris. The addition of isopropanol to the solution precipitates DNA solution from RNA and DNA is separated via centrifugation.
The binding of DNA to silica under high-salt conditions forms the basis of the technique. Chaotropic salt like guanidine hydrochloride disrupts cells, deactivates nucleases, and permits nucleic acids to bind to silica. RNase addition ensures the binding of pure DNA
Washing buffers generally consist of alcohols and they can be used for removing salts, proteins, and other contaminants from the upstream binding buffers or the sample. In addition, alcohol aids in the association of nucleic acid with the matrix.
In low ionic-strength buffers like nuclease-free water and TE buffer, DNA is soluble. When an aqueous buffer is applied to the silica membrane, DNA is released from silica, and eluate is collected. The purified and high-quality DNA is ready for use in various downstream applications like transfection, sequencing, multiplex PCR and coupled in vitro transcription/translation systems.
During the selection of the elution buffer, it is significant to consider the requirement of downstream processes. As long as EDTA does not impact chosen downstream application, elution and storage of DNA in TE buffer is helpful. EDTA binds or chelates with magnesium present in the purified DNA and this aids in inhibition of contaminating nuclease activity. If EDTA is a concern and the acidic nature of DNA leads to autohydrolysis, 10 mM Tris-HCl, 0.1 mM EDTA (pH 8) buffer can be used for the storage of DNA.
A short introduction on DNA and its significance have been provided. A gist on methods used so far for the isolation of DNA has been given. The basic steps involved in the isolation have been discussed. At the same time, five steps in the isolation of pure DNA have been detailed.