Compiling The Workings Of DNA Electrophoresis

Abstract

This paper will discuss the methods and applications of gel electrophoresis. This researcher will address the production of a gel from agarose and a buffer solution. The role of the gel, the buffer solution, and the intricacies of the electrophoresis machine will be capitalized upon. The laboratory experiences of this researcher in extracting DNA from plant-based materials will be synthesized with various articles that will assist in forming a working definition of DNA electrophoresis.

Electrophoresis is prized in the fields of biology and forensics for its ability to separate particles of DNA by their mass. This is achieved by placing raw DNA into an electrophoresis chamber containing both a specific semi-solid material resistant to the movement of the negatively charged DNA particles to the chamber’s (positively charged) cathode and a salt and water buffer solution that is conducive to an electric current. This researcher applied the practices of DNA electrophoresis as scientific support for the extraction of DNA from a peach and from wheat germ samples. However, to further delve into the materials and processes necessary to develop a deeper understanding of electrophoresis, an analysis of the roles of the gel, buffer, and electrophoresis chamber and power source will also be addressed.

The Process of Extracting DNA

The purpose of extraction is to obtain a sample of DNA that can then be inserted into the electrophoresis chamber. These DNA particles will then be essentially filtered into partitions based on mass. To do this, the organic plant material being used, the cell, and finally the nucleus must be broken down to free the contained DNA. This researcher selected a peach to be the fruit from which DNA would be extracted.

A solution of Dawn dish soap, water, and salt was mixed in the lab; the soap functions to break down the lipids of which the cell wall is composed, and the salt assists the DNA in aggregating. One hundred milliliters of the soap solution, as well as the peach sample, was placed in a processor and blended on high for 1 minute, simultaneously slicing down the organic plant material and allowing the soap solution to break down the cell walls. The next step in extraction is filtering the blended peach mix through a very fine material, such as a coffee filter. Filtering gave the researcher a solution devoid of larger fruit chunks but containing miniscule packages of DNA. To this solution, the researcher added twenty-five milliliters of meat tenderizer. The role of meat tenderizer in DNA extraction is similar to its role in softening meat by cutting apart the large meat proteins. The addition of meat tenderizer severs the proteins that bind DNA into its tight modules, allowing it to unspool into a more visible and collectable state. A quantity of meat tenderizer solution is pipetted into a test tube. This sample is collected primarily from the surface, as a solution without meat tenderizer particles is more desirable, and as the heavier particles of meat tenderizer sink to the bottom of the solution. In the next step, chilled ethyl alcohol is gradually added to the test tube to create a layer atop the peach extract solution. It is in this boundary region between the alcohol and the peach extract solution that the DNA will rise and manifest as a cloud-like cluster of strands that can be collected via micropipette. The sample is placed into a micro-centrifuge tube and centrifuged for three minutes, during which time the DNA coalesces into a pellet. The pellet must air-dry, then it is frozen overnight after the addition of fifty microliters of buffer. The buffer and pellet should be thawed and mixed after removal from the freezer, at which point the DNA is ready for insertion into the wells of the gel within the electrophoresis chamber. It must be ensured that the DNA is pipetted directly into the well--if the well is blown out, or if the DNA floats above the well in the buffer solution, banding in the gel will likely not occur.

Electrophoresis

Electrophoresis is achieved in a system in which an agarose gel is floating in a buffer that can conduct electricity. It can be determined if the chamber is functioning once turned on and adjusted to run at a certain voltage by the presence of bubbles rising at both ends of the chamber. During production, the gel is punctured with holes, or wells, in which the DNA will be inserted. When the chamber is activated, current begins to flow through the gel, and the principles of magnetism (i.e. “opposites attract”) begin to apply as the negatively charged DNA particles begin migrating towards the positive electrode (Gel electrophoresis). Although this researcher’s experiment did not require an established measurement standard for the DNA particles being sorted, in other analyses a DNA ladder may be used. Inserted into a well adjacent to the researcher’s DNA, DNA ladders “contain DNA fragments of known lengths. Commercial DNA ladders come in different size ranges, so [the researcher] would want to pick one with good 'coverage' of the size range of [the] expected fragments” (Gel electrophoresis).

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Behavior of DNA. The value of the charge of a DNA particle has a linear relationship to that of its mass. The gel, being viscous and microscopically porous, allows for smaller particles to travel through it at a greater velocity than the larger particles. The mass of DNA particles is determined by the length of the strands; once the electrophoresis chamber has run for the allotted time, the DNA samples should have created a banding pattern due to the varying speed and ease of migration through the gel. As DNA is opaque, the banding patterns can be difficult to see. This issue was remedied in this researcher’s experiment by injecting UView dye into the DNA and buffer solution prior to micropipetting this mixture into the wells of the gel. As implied by the product name, UView allows the banding to become highly visible when exposed to ultraviolet light.

Role of Gel. The gels used in DNA are created by mixing agarose, a material “isolated from the seaweed genera Gelidium and Gracilaria” (Lee, Constumbrado, Hsu & Kim 2012) with a buffer solution. The ratios of agarose to buffer determine the concentration percentage of the gel. This resulting mixture is heated to boiling, then stirred and cooled to a temperature of between 50 and 55 degrees Celsius before being poured into a mold. The gel formed possesses a chemical structure that allows it to have numerous microscopic pores through which the DNA particles will travel when stimulated by electric currents (Gel electrophoresis). A comb placed at a certain region of the gel mold creates the wells for DNA injection.

The Electrophoresis Chamber. The electrophoresis chamber is a small bin with an elevated center portion on which the gel will be placed. A positive (cathode) and negative (anode) electrode are placed at opposite ends of the chamber. The same buffer solution used to create the agarose gel is poured into the electrophoresis chamber to a level at which it just covers the gel. In anticipation of DNA’s direction of travel, the end of the gel containing wells should be placed at the negative side of the chamber. The cover of the chamber is wired to a power source that allows the user to dictate the running time and the voltage. As stated previously, once the chamber is running, the DNA will naturally begin moving towards the positive pole--the values at which the power source is set is at the operator’s discretion. However, DNA electrophoresis is best performed at around 120 Volts, and if left running for too long, the DNA sample will migrate completely through the gel.

Applications of DNA Electrophoresis

The purpose of this researcher’s use of DNA Electrophoresis was verification of the achievement of DNA after the performance of a peach DNA extraction procedure. However, electrophoresis has multiple purposes, particularly in the scientific fields of botany, forensics, and genetics. Some of the various uses of electrophoresis were specified by Matthew Robbins in his paper “Gel Electrophoresis Principles and Applications” as “[visualizing] bands of a molecular marker to genotype individual plants, [verifying] amplification by PCR or sequencing reactions [and separating] DNA fragments to clone a specific band” (2018).

Conclusion

The process and results of DNA electrophoresis are made possible by multiple factors, including the proper extraction and isolation of DNA, the agarose gel and buffer solution, and the operation of the electrophoresis power source. While this researcher only applied electrophoresis for confirmation, the uses are diverse and multidisciplinary. Avenues of further study are doubtlessly still open when it comes to this method of isolating specific masses of DNA.

References

1. Gel electrophoresis [Khan Academy]. (n.d.). Retrieved from https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/gel-electrophoresis.
2. Lee, P.Y., Constumbrado, J., Hsu, C. & Kim, Y.H. (2012). Agarose gel electrophoresis for the separation of DNA fragments. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846332/.
3. Robbins, M. (2018). Gel electrophoresis principles and applications. Retrieved from https://articles.extension.org/pages/32366/gel-electrophoresis-principles-and-applications.