# Chapter 4: How do you work with DNA?

Genetic information is stored in the molecule of DNA, the properties of which were described in the previous chapter (Chapter 3 - Meet DNA). In this section, we present some of the most used methods of DNA analysis, which are applied not only in basic research, but also in the diagnosis of diseases, genealogical studies, crime investigation, food control, and other fields.

If we want to study the DNA molecule, we must first obtain it from the cells. We call this process DNA isolation or purification. The first isolation of DNA was performed (albeit unintentionally at the time) as early as 1869, long before its spatial structure was discovered. Friedrich Miescher, a Swiss physician, took leukocytes (a type of immune cell) from bandages he used to dress his patients' wounds and wanted to focus on isolating various proteins. In his experiments, he was interested in a particular substance isolated from the cell nucleus that, unlike proteins, did not contain sulphur. This substance precipitated under acidic conditions, while it dissolved under alkaline conditions. Since it came from cell nuclei, he called it nuclein (the name nucleic acid was introduced later by his student Richard Altman).

Since DNA is located inside cells, the first step in DNA isolation is cell disruption or lysis. Cells have a cytoplasmic membrane on their surface, and some also have a stronger cell wall (e.g., plant cells, yeasts, bacteria). We need to break these surface structures to release the cell contents into solution. In principle, there are 3 approaches to breaking cells, and they are usually combined. We can break up the cells mechanically, e.g., by rubbing them in a rubbing bowl or shaking them with special glass or ceramic beads. Mechanical stress on the cell surface is also achieved by ultrasound and sudden temperature changes by freezing and thawing. The so-called digestion of the cell wall is made possible by enzymes isolated from various organisms, e.g., zymolyase isolated from bacteria cleaves the cell wall of some yeasts, lysozyme isolated from saliva or tears cleaves the cell wall of bacteria, and cellulase isolated from some fungi cleaves the cell wall of plant cells. In addition, there are chemical substances that destroy the integrity of the cell surface, and among them the most used are various detergents since lipids are the main component of the membrane. Most laboratories use sodium dodecyl sulphate (SDS) or Triton X-100 as detergent, but any detergent will suffice for DNA isolation outside of laboratory.

Nucleic acid is naturally surrounded by a variety of proteins, lipids, sugars (polysaccharides), and other substances that remain in contact with it even after cell lysis. Separation of DNA from these components is the next important step in DNA isolation and is referred to as DNA extraction. Efficient DNA extraction is possible thanks to the different chemical properties of each molecule surrounding the DNA. Detergents, as mentioned earlier, are used to break down the remnants of membranes, including the nuclear membrane and other lipid structures. Large amounts of proteins in the cell can be precipitated and separated with organic substances - for example, a mixture of phenol, chloroform, isoamylalcohol, or guanidine chloride. After mixing the sample with phenol and chloroform or with chloroform and isoamylalcohol, two layers are formed. The lower layer is organic, in which dissolved impurities remain, i.e., the unwanted components such as lipids and parts of proteins. A protein ring forms between the two layers, and above this is the upper, aqueous layer, which contains solubilized DNA, usually together with RNA. A major obstacle in isolating nucleic acids is the presence of nucleases - enzymes that degrade nucleic acids in cells. Most nucleases are localized in lysosomes, but when the cell is disturbed, they are released (lysosomes also disintegrate) and therefore pose a threat to the isolated DNA. Suppression of nuclease activity is usually achieved by addition of anionic detergents or EDTA (ethylenediaminetetraacetic acid), which absorbs divalent ions (mainly Mg2+) that serve as nuclease cofactors.

After removal of the aqueous phase, which also contains DNA, the DNA must be precipitated from the solution to successfully purify and isolate it. Pure ethanol or isopropanol is used for precipitation as the addition of alcohol changes the properties of the nucleic acid making it less soluble in water. Precipitation is also supported by the addition of monovalent ions (usually Na+), so sodium or ammonium acetate is added to the mixture. After centrifugation, the precipitated DNA settles, and the remaining alcohol must be thoroughly removed and the DNA dried. Since the precipitated DNA is very often isolated together with RNA, we dissolve the nucleic acids and incubate them with the enzyme RNase, which only cleaves RNA molecules. Then, the DNA must be repeatedly precipitated to get rid of the RNase and the excess solution. Ethanol or isopropanol at low temperature is used for this step. After centrifugation, we need to thoroughly remove the alcohol, because even the residual amount of ethanol could interfere with the subsequent reactions in which we want to use the DNA. Finally, we can dissolve the prepared DNA in water.

Because DNA isolation is often a necessary step for further analysis, many companies have developed commercial kits for their isolation to speed up and simplify the whole procedure and avoid working with chemicals that are hazardous to health, such as phenol and chloroform. However, the principle of such isolation is not much different from the one which has just been described.