The advent of DNA research has come to such heights that the topic has been the focus of many fields. Since its conception in the 1800’s, DNA has been implicated in hundreds of applications, including identification, genetic screening, testing, drug development and other therapeutically inclined fields. To be successful in these endeavors, it is a crucial prerequisite to learn how to extract high quality DNA and keeping it clean, purified and intact. This experiment demonstrated a simple way to do this using common household equipments.
Peas were selected as the host, and were blended with a salt solution to release the cells. The cells were then lysed using detergent, after which the proteins were degraded using meat tenderizer powder. Alcohol was then added, precipitating the DNA out of the water layer. The acquire DNA was of good quality, although low in total yield. This could have resulted from several reasons, such as contaminations, DNA denaturation, and mechanical disruption.
Future repetitions of this experiment should be done in a cleaner environment, keeping track of the said factors to optimize the extraction procedure of the DNA. Introduction Probably one of the most important scientific breakthroughs in the 19th century is the discovery of the blueprint of life, the deoxyribose nucleic acid, or simply the DNA. It was through the combined efforts of several scientists that we today have gained a lot about this molecule and are able to perform several studies about it.
This all began when Friedrich Miescher discovered a substance called ‘nuclein’, followed by the uncovering of the different possible components of it (Dahm, 2005). The next big breakthrough was when two groups of scientists – one led by Frederick Griffith and the other by Oswald Avery – proved that DNA indeed was the “transforming principle” behind the alteration of a smooth bacterial trait to a rough Pneumococcus. After mixing killed smooth bacteria with live rough ones, the latter surprisingly transformed into their smooth counterpart (Lorenz and Wackernagel, 1994).
Later on, Alfred Hershey and Martha Chase performed their classical experiment, showing that DNA is passed on by a phage upon infection, verifying early assumptions of the DNA being the genetic material of all organisms (Hershey and Chase, 1952). The importance of the DNA can be explained by the central dogma of molecular biology, which defines the exact role of the DNA in the body. The process begins at the DNA, where the genetic information of the host organism is kept. DNA is capable of replicating itself, creating an exact copy while retaining the original one.
This molecule is then transcribed to form the RNA, or the ribonucleic acid, which is then translated into proteins as suggested by initial genetic information. The proteins and other side products would then define the entirety of the host organism – from its physical appearance to its body’s processes, and so on (Crick, 1970). Today, the study on DNA continues as many hidden treasures are continually discovered. One example would be the recently concluded Human Genome Project, where a complete map of the human’s genome, or the entire genetic content, is made available for disposal.
This provides a huge backdrop on which many fields can gain advantage to. One possibility is on the therapeutic and medical areas, where diseases can better be understood by studying the portion of the DNA directly causing the disorder. Drugs can then be easily developed, targeting the products produced by these faulty DNA pieces. Another possible option would be to directly modify the affected gene. The importance of DNA also traverses the plains of identification, where the molecule becomes a unique identifier of its host.
This is crucial in surveillance and crime scenes, where the DNA from small traces of body debris such as fluids, skin and even hair can be extracted and directly matched with possible suspects or victims. Identification also has its share in parental testing. Since DNA is hereditary, it is possible to compare samples from the child with the adults in question, and more than always, the true parent would resemble similarities with the offspring’s DNA. These are just a number of the possible purposes of DNA, and many more in archive and waiting just to be discovered.
In order to efficiently study DNA, one must first be able to extract the molecule from the cells containing it. It should be done with care as not to degrade the molecule and completely isolate it from other celluar debris. Through the years, several methods have been employed by different scientists, and today several biological companies have created “kits” containing their own concoctions and chemicals for DNA extraction. In this experiment, DNA would be extracted from green peas using a method familiar to the scientific community – the alcohol precipitation method.
This exploits the fact that DNA precipitates and can be retrieved under alcoholic conditions while contaminants remain dissolved (QIAGEN, 2008). The here twist lies in using home-ready materials and equipment in performing the experiment. Materials and Methods A half cup containing about 100 ml total volume of selected peas was first washed to remove adhering dirt and contaminant. Together with 1/8 teaspoon salt and 200 ml cold water, the solution was blended on high for 15 seconds.
The solution was then filtered through a strainer into a clean glass jar. Two tablespoons of detergent was then mixed in, and the resulting solution was allowed to sit for about 10 minutes before transferring to a smaller jar. A pinch of meat tenderizer powder was added afterwards. After swirling the mixture, about 2 ml of 95% ethyl alcohol was slowly poured in. The extracted DNA, observed as white substances on the interface between the alcohol and the water layers is then obtained using a thin stick, and then stored in alcohol. Results and Discussion
There are basically three parts in DNA extraction – releasing the genetic material, isolating it from other unwanted debris, and then purifying it for storage and use. In our experiment, the cells were disaggregated released by continuous blending of the peas. It is important to note that blending can be harsh for some samples, such as animal cells. Blending for a long time can destroy the cells, including the DNA contained inside. Plant cells, having a stronger cell wall, can withstand more mechanical stress such as that provided by the blender (Campbell and Reece, 2008).
It is also important to add salt to the mixture before blending. Salt creates an isotonic solution, preventing premature lysis of the cells upon releasing them after blending, for we want to extract the DNA later on. The thin mixture is then strained to remove the large debris. Detergent, known to disrupt cell membranes, is then added to the filtered solution. These surfactants lyse the cells, or bursts them open, releasing their contents into the solution (Campbell and Reece, 2008). Enzymes, such as meat tenderizers are then added to cut the long proteins and other unwanted cellular components.
Alcohol is finally added to the mixture. The result would be a two phased-solution, an upper organic (alcohol) layer and a bottom aqueous (water) layer. Since alcohol is soluble in water, the alcohol would cause the DNA to precipitate and come out of the solution, just at the separation point between the two phases (Campbell and Reece, 2008). The DNA can then be carefully fished out using a stick or a cotton bud. The extracted DNA from the experiment was a white, stringy substance. Although obvious to be the expected product, the yield was actually small.
This can be attributed to a number of reasons. Firstly, it is possible that the DNA got denatured and destroyed during extraction. Long exposure to mechanical stress might have cut up the DNA, resulting to shorter and smaller strands. The temperature of the environment should also be taken into consideration. DNA tends to be denatured at high temperatures, so it is best to keep the solution cool at all times. Another possible cause is the enzymes added. It is plausible for the enzymes to interact and affect the DNA together with the other proteins.
Finally, contaminants may have entered the system, such as DNAses, which are enzymes that can degrade DNA instantaneously (Campbell & Reece, 2008). It is then recommended to do the experiment under sterile conditions, keeping all equipment as clean as possible. The other factors, such as temperature and length of blending, should also be adjusted and optimized for better results. Conclusion DNA extraction using the alcohol-precipitation method can be done using simple household materials. The chance of obtaining the molecule is pretty high, but is usually compensated by a low amount of yield.
Several optimizations should be made on the procedure, taking note of several factors that could have degrade or destroy the required molecule. Though not done in this experiment, purification of the DNA should also be commenced right after extraction, since alcohol-precipitation methods are very much prone to remaining protein contamination.
References:
Campbell, N. A. & Reece, J. B. (2008) Biology. Benjamin and Cummings:CA. Crick, F. (1970). “Central dogma of molecular biology”. Nature. 227: 561-563. Dahm R (2005).”Friedrich Miescher and the discovery of DNA”. Dev Biol 278 (2): 274–88. Hershey A, Chase M (1952). “Independent functions of viral protein and nucleic acid in growth of bacteriophage”. J Gen Physiol 36 (1): 39–56. Lorenz MG, Wackernagel W (1994). “Bacterial gene transfer by natural genetic transformation in the environment”. Microbiol. Rev. 58 (3): 563–602. QIAGEN. (2008). “DNA isolation methods”. QIAGEN website. Retrieved November 25, 2008 from http://www1. qiagen. com/literature/brochures/Gen_DNA_Pur/1019469_BROS_DNYTi_p10_13. pdf