DNA is composed of three parts- a phosphate group, a 5-carbon (pentose) sugar called deoxyribose, and a nitrogenous base. In DNA, there are four kinds of nitrogenous bases. These bases are called adenine (A), thymine (T), cytosine (C), and guanine (G). DNA is a negatively charged particle, and its negative charge comes from the phosphate component. “The backbone of DNA is formed by sugar- phosphate- sugar chain. Phosphate groups in the DNA backbone carry negatively-charged oxygen molecules giving the phosphate-sugar backbone of DNA an overall negative charge.” (Rani, 2019). In each cell, the DNA is located inside of the nucleus with a small amount found inside of the mitochondria. This is why extraction is such a complex process. To get to the DNA inside the nucleus, the rest of the cell must be broken down. The cell wall, the cell membrane, and the nuclear membrane all must be destroyed to release the DNA.
DNA extraction is the process that is used to isolate DNA (deoxyribonucleic acid) from a biological sample using various chemical and physical methods. This method is very important in biotechnology, as it is used in everything from research to making diagnostic and therapeutic decisions. It is also used in forensics and molecular biology. Extraction is also important in determining characteristics of DNA such as size, shape, and function. Extraction of DNA is also extremely important in studying genetic causes of disease, and in developing drugs.
DNA was first extracted in 1869 by Swiss physician, Friedrich Miescher. Meischer first extracted DNA as part of an experiment in which he hoped to determine the chemical compositions of cells. “In the course of his work on leucocytes he noticed the precipitation of a substance when acid was added and that this dissolved following the addition of alkali. Miescher decided to call the new substance 'nuclein' by virtue of its presence in the nuclei of the cell. Upon further analysis, Miescher noted that the chemical composition of the substance differed from proteins and other known molecules.” ('DNA extraction isolates DNA from biological material', 2019). Since then, many advances have been made in DNA extraction. This method used to be very complex and intense, and made very little DNA. Now, extraction has many specialized methods.
In the experiment done in class, the fruit used was pineapple. To start, the pineapple was put in the microwave for 30 seconds to defrost. After it was defrosted, the pineapple was then placed in a mortar and pestle and ground into a smooth liquid. Then, the fruit liquid is added to a solution containing 1 mL of liquid detergent, 1 gram of salt, and 20 mL of hot tap water. This solution was used to break down the cell wall so that the DNA could be released. The contents were stirred constantly for 5 minutes and then poured into a test tube with a lid. The tube was then placed in a 60° Celsius water bath for 10 minutes. After the water bath, the mixture was poured into a glass test tube. 1 milliliter of alcohol was added to the test tube creating a layer on top of the pineapple mixture. This is done by slowly releasing the alcohol down the side of the test tube while holding the tube at an angle, making sure not to mix the two layers. The tube is allowed to sit undisturbed for a few minutes until DNA begins to form between the two layers. Once the DNA forms, it is removed with a micropipette and placed into a 1.5 mL microcentrifuge tube. The tube is then placed in the centrifuge at 1300 RPM for 2 minutes. The tube is centrifuged to separate the DNA from any excess liquid that may have been picked up when the DNA was pipetted from between the two layers in the glass test tube. After centrifuging the tube, there should be a small pellet of DNA in the bottom of the tube. Pour out the excess liquid from the tube while keeping the pellet in the tube, and allow the pellet to air dry. After the pellet has dried, 50 microliters of TE buffer is added to the tube and then the tube is placed in the freezer until it is time to run the DNA in the electrophoresis.
Electrophoresis is used to separate particles according to size, charge, and shape. Inside the electrophoresis chamber there is a gel made from agarose and buffer. The chamber is filled with buffer and the gel is placed in the buffer. Since DNA has a negative charge, when it is run in an electrophoresis the wells on the gel must be at the negative end of the chamber. This is done so that the negatively charged DNA can run towards the positive end of the gel. Particles are moved along the gel as an electric current is passed through it. “An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge. The gel consists of a permeable matrix, a bit like a sieve, through which molecules can travel when an electric current is passed across it. Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance.” (Austin, 2019). Because of this, particles are separated by size. The separated particles are what forms the bands that are seen on a gel.
The electrophoresis can be adjusted according to what is being run in the gel. The electrophoresis box is connected to a power supply that plugs into the wall. Electrophoresis power supplies can usually run at least four electrophoresis chambers. On the power supply, there are buttons to adjust the time that the gel is run, and the amount of volts run through the gel. If the gel is allowed to run for too long or at too high of a voltage, the DNA can run off of the gel. The gel can also get too hot and melt if the settings are too high.
When running the DNA that was extracted in the experiment done in class, a loading dye called UView was added to the DNA. To do this, the frozen microtubes were allowed to warm up until they were no longer frozen. Ten microliters of the DNA were added to a new, clean microcentrifuge tube using a micropipette. Then, two microliters of UView loading dye were added to the tube as well, using a new micropipette tip to avoid contamination. The tube was then vortexed until the DNA and the loading dye were mixed. Once they were mixed, the solution was inserted into a gel. To insert the samples into the gel, a micropipette set at 12 microliters was used. When inserting the DNA, it is important to make sure that the micropipette tip does not break through the bottom of the well, and make sure that the tip is far enough in the well so that the DNA doesn’t disperse throughout the chamber. After the DNA is inserted into the well, the power supply needs to be set. For this experiment, the power supply was set at 120 volts, and run for 15 minutes. After pressing the ‘run’ button on the power supply, there should be bubbles rising from the electrodes at each end of the chamber. Once the electrophoresis has finished running, there should be bands formed on the gel. The different bands show the different particles that were contained in the DNA. The loading dye was mixed with the DNA to help the bands be more visible. Without the dye, the DNA would have been a clear color and extremely difficult to see.
Works Cited
DNA extraction isolates DNA from biological material. (2019). Retrieved from https://www.whatisbiotechnology.org/index.php/science/summary/extraction/dna-extraction-isolates-dna-from-biological-material
Austin, C. (2019). What is gel electrophoresis?. Retrieved 6 November 2019, from https://www.yourgenome.org/facts/what-is-gel-electrophoresis
Rani, A. (2019). Why does DNA possess a negative charge due to phosphate groups? - Quora. Retrieved 7 November 2019, from https://www.quora.com/Why-does-DNA-possess-a-negative-charge-due-to-phosphate-groups