DNA extraction (Microorganism, blood, cell lines, Tissues)

The foundation of all biotechnology research is the modification and analysis of DNA. Before any other techniques, such as recombinant DNA preparation and diagnostic tests, researchers need methods to extract DNA from various organisms and purify it to a certain extent. The techniques used to extract DNA vary based on the source of the sample, its size, and its age.

Due to the diversity of these methods, there are common points among them, which involve separating DNA from the other cellular components. This DNA can be chromosomal, plasmid, or viral, and it can be extracted from all biological samples such as living and preserved tissues, cells, and more. The DNA extraction process must be performed as gently as possible to avoid damaging the DNA and breaking it into smaller fragments.

DNA was first extracted in 1869 by Friedrich Miescher, a Swiss physician who attempted to isolate cells from lymph nodes for his research. However, obtaining an adequate concentration of lymphocytes was very difficult, so the targeted cells were changed to white blood cells obtained from pus collected in surgical dressings.

Initially, Miescher focused on the different types of proteins that make up white blood cells. He was able to demonstrate that proteins are the main component of the cytoplasm. During these experiments, he observed that when acid was added to the solution, a substance precipitated, and when a base was added, it dissolved again. In this way, the first precipitation of contaminants resulting from DNA was obtained.

DNA Separation

Friedrich Miescher designed a new protocol for separating DNA from the proteins present in cell extracts. This protocol was designed to separate the nucleus from the cytoplasm, and then extract the DNA. The first protocol was unable to produce a sufficient quantity of product. A second protocol was needed to prepare larger amounts of pure nucleic acids, which were later named nuclear acid by Richard Altman.

After Miescher, further efforts led to significant advancements in the DNA extraction process. The first routine laboratory procedures for DNA extraction were developed based on density-gradient centrifugation strategies. Meselson and Stahl used this method in 1958 to explain the semi-conservative replication of DNA.

Subsequent procedures benefited from the differences in solubility of large chromosomal DNA, plasmids, and proteins in alkaline buffers. Now, many dedicated methods for extracting pure DNA have been established, and these methods are often available in the form of commercial kits.

Genetic Analysis

Extracting DNA for genetic analysis for scientific, medical, and forensic purposes is essential. Scientists use the extracted DNA in cases such as introducing foreign DNA into specific cells or in diagnostics. In the medical field, this process holds diagnostic value. In forensic matters, extracted DNA is used to identify individuals. The sources of DNA used can be highly diverse and can be obtained from any living or non-living organism. Common sources include whole blood, hair, sperm, bones, nails, saliva, epithelial cells, urine, and bacteria.

Thus, it is clear that each method of DNA extraction must be tailored to the target sample so that the DNA can be effectively separated from the relevant sample. The next issue is the sample size. For example, the extraction method for a small sample like sperm or a single hair differs from the method used for several milliliters of blood.

Another important factor is the age of the sample. The sample can be fresh or preserved. Preserved samples may come from blood or frozen tissues, or from bones or archaeological samples from humans, animals, and plants.

The easiest extraction process is extracting DNA from bacteria because bacterial cells do not have many structural components beneath their cell membrane. Bacteria such as E. coli are the chosen organisms for modifying all kinds of genes due to the ease of extracting DNA from them. E. coli contains both genomic and plasmid DNA, with the difference being that genomic DNA is much larger.

Cell Lysis

The DNA extraction process typically begins with the lysis or disruption of tissues and cells. For this, the cell membrane must be destroyed. This process is essential for breaking down protein structures and releasing nucleic acids from the nucleus. Generally, the sample is lysed in a saline solution containing detergents and proteases such as Proteinase K. This process leads to the breakdown of cellular structures and the dissolution of the membrane. In bacteria, an enzyme called lysozyme digests the peptidoglycan that forms the main structure of the cell wall. Then, a detergent such as sodium dodecyl sulfate (SDS) destroys the membrane by disrupting the lipid bilayer structure.

When extracting DNA from other organisms, the method of cell destruction depends on their structure. Tissue samples from animals and plants must be cut into smaller pieces to release the compounds inside the cells. Plant cells must first be thoroughly broken down in a blender, which breaks the rigid cell walls. The tissue walls are then digested with enzymes, which helps convert the long polymers of lignin and cellulose into monomers.

DNA is extracted from animal tissues, such as mouse hair samples, after the tissues are broken down using Proteinase K and the membrane is dissolved using detergents. Cultured cells are the easiest cells from which to extract DNA, as they do not have a cell wall or other structures outside their cell membrane. What the detergent does is simply break down the membrane to release the compounds inside the cells. Each living organism and tissue requires some slight modifications in the procedure to release compounds like DNA from within the cells.

Cell Disruption

Disrupting soft tissues and cells in DNA extraction is easy. However, sometimes we need to do this with hard tissues such as bones and woody plants. Many woody plant samples are frozen in liquid nitrogen and then immediately crushed into powder. Bones contain many mineral materials, and ions must be removed before extraction so they do not interfere with processes such as PCR. After these treatments on the sample, a homogenization process must be performed using appropriate devices to make the mixture homogeneous. Then the lysis process is performed.

After releasing the compounds inside the cells, they must be separated from the remnants of the cell membrane, bones, cartilage, cell walls, and others using centrifugation or chemical extraction methods. Centrifugation leads to the separation of particles based on their size, with larger and heavier molecules precipitating faster than smaller ones. Additionally, insoluble materials in the aqueous phase form clumps that quickly settle at the bottom of the centrifuge tube. For example, after breaking the cell wall, the resulting pieces become smaller than the DNA molecules. Centrifugation aggregates the DNA molecules into a pellet while the soluble fragments of the cell wall remain in the tube.

Separation of Cell Fragments

Another method used to separate cell fragments is chemical extraction using phenol. This method is used to separate unwanted proteins from DNA. Phenol is an acid that can destroy 60 to 70% of biological molecules, including proteins. Phenol is not water-soluble, and after mixing it with an aqueous sample containing DNA and protein, two mixed layers are separated from each other. The protein remains in the phenolic layer, and the DNA is in the aqueous layer. The two layers are separated by centrifugation, and in this way, the DNA in the aqueous layer is separated from the phenol.

After separating DNA molecules from proteins, the sample still contains both RNA and DNA types. Since RNA is also a nucleic acid, it does not dissolve in phenol. An enzyme called ribonuclease (RNase) converts RNA into ribonucleotides. As a result, a sample of DNA is produced that exists in a solution containing RNA fragments and ribonucleotides. When an equal volume of alcohol is added, a very large piece of DNA is separated from the aqueous phase and isolated by centrifugation, while the ribonucleotides remain in solution. The resulting DNA can be used in various experiments.

Plasmid

In genetic engineering, three types of DNA are typically extracted:

• total DNA present in the cell

• plasmid DNA

• bacterial viral DNA.

Total DNA is often used as a source for gene preparation for cloning purposes and includes the genomic DNA of the organism along with other DNA molecules present, such as plasmids. The method for extracting plasmid DNA is similar to that for extracting total DNA, with the difference that at one stage, the plasmid must be separated from the other DNA molecules.

Plasmid DNA is frequently extracted in modern molecular biology processes when required as vectors. Plasmid DNA is usually extracted from viral particles, not from bacterial cells infected with the virus. However, there are exceptions. In this case, special techniques must be used to remove the capsule.