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Deoxyribonucleic acid is abbreviated as DNA, which is a complex molecule present in the nucleus of a cell. It is the hereditary material in organisms, containing all the information necessary for building and maintaining an organism. DNA is composed of two polynucleotide chains, which coil around each other, forming a double helix, thereby carrying genetic information for reproduction, growth, functioning, and development of organisms.
Extraction of DNA is a significant phenomenon as the extracted DNA is used in many applications and fields like science, medicine, detection of microbes, sequencing of genomes, determination of paternity, and forensic science. DNA acts as a forensic tool for the identification of war victims, accidents, thieves, and rapists. DNA paternity test and recombinant DNA technology are quite popular. Hence, it is a requisite to understand the principle of extraction of DNA so that the methodology can be carried out accurately to obtain quality material with a higher yield. To enlighten the readers with the concepts, this article brings about the fundamentals involved in the extraction of DNA.
DNA is one of the complex biomolecules, consisting of the genetic code with the instructions for the functioning and development of all living organisms, except some viruses. The DNA segments, which carry the genetic information are called genes. DNA is transcribed into RNA and used as a template in protein synthesis.
Sources of DNA
The process of extraction of DNA leads to the isolation of DNA in a pure form. The extraction process is a preliminary step, where purified DNA is obtained among lipids, proteins, and RNA among the cellular components. DNA is isolated from nucleated cells from various sources like blood, urine, bacteria, epithelial cells, tissues, plants, faeces, nails, bones, shed feathers, egg-shells, saliva, sperm, and hairs from both living and dead cells.
Though there are numerous methods for the extraction of DNA, they all start with the lysis of cells. Then, it is followed by deproteination and DNA recovery. Differences between the approaches lie in the extent of deproteination and the molecular weight of the extracted DNA. Age, sources, and size of the samples are the factors, which affect the method of DNA extraction.
Initially, the cell membrane is disrupted physically or chemically to obtain a fluid consisting of all the components including DNA and RNA. This process is known as cell lysis and the resulting fluid is called lysate. During cell lysis, various reagents and chemicals are used to break down distinct cell components. Proteins are broken down by protease, lipids are broken down by surfactants and detergents, and RNA is broken down by RNase.
The obtained lysate is treated with a concentrated salt solution to make broken components clumped together, leaving the DNA to float freely in the solution.
The solution containing lysate, surfactants, detergents, lipids, RNA, and broken proteins is centrifuged for the separation of clumped debris from DNA.
Finally, precipitation of DNA is performed by the addition of ice-cold alcohol and salt for increasing the ionic strength, which enhances the precipitation process. Upon centrifugation of the solution, a pellet of DNA is obtained. The pellet is stuck to the wall of the Eppendorf, while the supernatant is discarded. The obtained pellet is suspended either in a slightly alkaline solution, namely TE buffer or ultra-pure water for the removal of dissolved gases and organic particles. The suspended DNA is used for further experimentation like amplification by PCR.
For studying DNA, it needs to be taken out of the cell. DNA is organized as a chromosome in the nucleus in most eukaryotic cells like human and plant cells. Since bacterial cells possess no nucleus, DNA is organized in circular plasmids or rings. The process of extraction of DNA frees DNA from the cell and helps in separation of DNA from cellular fluid and proteins, so finally there is some pure DNA left.
Lysis of cells and tissues destroys the protein structures and allows the release of nucleic acids from the nucleus. The lysis is achieved using a lysis solution, namely sodium chloride, providing an osmotic shock to the cells. Tris HCl is a buffer to retain constant pH. EDTA sequesters the divalent metal ions, which are required for nuclease activity. A detergent, namely SDS is used to disrupt the cell membrane and nuclear envelope, which causes the cells to burst open and release their respective DNA. Now, DNA is still wrapped tightly around histone proteins. Proteinase K is a serine protease enzyme, commonly used in DNA extraction. It cuts apart the histones to free DNA and results in the breakdown of cells by dissolving the membranes.
From the protein-nucleic acid complexes, nucleic acids are purified via phase extraction. A mixture of organic solvents (phenol: chloroform: isoamyl alcohol) is added in the ratio 25:24:1. Phenol aids in the dissociation of proteins from DNA. Chloroform helps in maintaining the separation of aqueous and organic phases through the denaturation of lipids and proteins. It reduces losses to the organic phase by making DNA less soluble in phenol. For the prevention of foaming, isoamyl alcohol is added. DNA partitions into the aqueous phase in the range of pH 7-8. Protein is denatured and extracted into a water-immiscible organic phase. It is separated from nucleic acid possessing an aqueous phase through centrifugation. A white precipitate is formed between aqueous and organic phases when a large amount of protein is present.
DNA is precipitated with isopropanol or cold ethanol after adjustment with 3M sodium acetate and centrifuged. As DNA is soluble in alcohol, it comes out of the solution. The alcohol acts as a wash for removing the residual salts and then, it is removed. Then, DNA is stored in a biological buffer, namely Tris-EDTA buffer. With the help of the enzyme RNase, contaminating RNA in the DNA sample is eliminated.
As shearing forces are generated in each step, the resulting DNA molecules in the final stage rarely exceed the length of 100 - 150 kb. However, DNA obtained in this size is enough for usage in southern blotting DNA analysis on standard agarose gels, template for PCR reactions, and construction of genomic DNA libraries in bacteriophage lambda vectors.
A short introduction to DNA has been provided. The significance of DNA has been given. A general outline of methodology has been shown. Then, an overview of each step has been briefed. Then, the justification of usage of every reagent and material used in each step has been discussed. Finally, the purification process and yield have been detailed.