Introduction
DNA, or Deoxyribonucleic acid, contains vital coding that makes up the entirety of an organism (Lesk, 2005). These long, double helix structures contain four nucleotides which sequentially create nucleic acids, then consequently combine in different ways to form specific proteins that perform various tasks for the organism during its lifetime (Sanderson, 2007). Once the animal reproduces, this genetic coding is passed on to offspring (Lesk, 2005). There are many practical uses for DNA extraction, which range from its use within forensic environments, (Brandt & Gonzales, 2005) to identifying the bloodline of an animal and its potential for mutations (Cocciolone & Timms, 1992). As each organism has a different and unique genetic code within their DNA; achieving a ‘match’ for unknown tissue has become a well valued process to understand more about animals and their genetic construct.
Students participated in two laboratories over the course of a week to extract DNA from four separate tissue samples from an unknown organism, one of which is known to be the target animal. The sample that matches with the specific target organism is anticipated to be discovered through utilising the processes of PCR, amplification, and gel electrophoresis.
Method
Prior to participating in laboratory one and two, standard hygiene procedures were utilised to minimise the effects of contaminating the samples. These procedures included washing hands thoroughly and using lab coats and safety glasses.
Materials and method for DNA extraction and PCR
Students first labelled a 2mL tube with their group name and the sample number of the tissue they had chosen. This tissue was cut, weighed to .25 grams and placed in the tube. 500μl of lysis buffer and 500μl DI water was then added, and the tube was gently flicked. 1 drop of 1% protease solution was then added using a transfer pipette. The large piece of tissue was split up using a tissue grinder until the solution was discoloured, and then vortexed for 10 seconds. The tissue samples were placed into an incubator for 10 minutes, and then students transferred the liquid of this sample ( with care to avoid solid tissue) into a centrifuge tube. 1mL of lysis buffer was added to the tube with the aid of a transfer pipette, and gently flicked. A new pipette was used to transfer 2mL of cold ethanol into the tube, at a 45° angle and immediately rested for 2 minutes. After 2 minutes, 200μL of DNA precipitate was then transferred to a 2mL tube, and 250μL of elution buffer was added. The PCR sample was then prepared by exchanging the DNA sample with one already filtered, purified and diluted. 20μL of DNA and 20μL of master mix was then added into a PCR tube, and immediately capped and placed on ice
Materials and method for Gel electrophoresis
PCR tube was placed into a small tube with lid cut off, then consecutively into a larger tube to fit centrifuge with other samples. The centrifuge was pulse- spun for a few seconds. 10μl of Orange G loading dye was loaded into PCR tube, and tube was centrifuged once more. The gel electrophoresis chamber and insertion of the allele ladder samples was done by lab demonstrators to ensure accuracy. One student from each group then placed 20μl of PCR sample into the chamber, and the well number was documented on each paper for identification purposes. For 30 minutes the electrophoresis chamber was run, at 100v. Following this process, the uv transilluminator was then used with the room in full darkness, and a photo was taken with a digital camera.
Results
These results identify the allele ladders present, followed by the positive controls in each chamber.
The 9 samples that are shown in the electrophoresis chamber are identified via their number, from one to four accordingly. In figure 2, both images also show a negative control.
Discussion
The role of DNA in science
DNA extraction is a process that involves the separation of strands of DNA from different parts of the cell (O'Sullivan, et al., 1999). This process is often utilised as a form of isolation or purification of the DNA prior to the PCR, or Polymerase chain reaction process. PCR is a synthetic process which replicates target sequences of DNA coding through periods of heating and cooling of Deoxyribonucleic acid until appropriate replications of the target DNA are created (Booth, et al., 2010). The target DNA at this stage is usually fabricated in extremely large quantities to ensure the efficiency of results (Reed, et al., 2007). Gel electrophoresis then occurs and is the process that pulls the DNA through smaller and smaller compartments with the aid of an electrical current until it cannot be pulled any further. These processes are frequently utilised in studies due to their diverse range of uses. For example, utilizing DNA proves useful in identifying blood or other fluids within a forensic setting (Ciampolini, et al., 2000), genetic analysis of animals in order to identify certain lineages or individuals (Cocciolone & Timms, 1992), and agricultural analysis and genetic alteration of crops (Brandt & Gonzales, 2005). Genetic diseases and cancer can also be diagnosed through applying these practices (Reed, et al., 2007).
Discussion of results
Upon completion of gel electrophoresis, the tissue sample number three matched with that of the positive control. This is easily identified due to the close match with that of the control sample in the results, as illustrated in figures 1 and 2, and clearly supports the original hypothesis. The tissue samples that were utilised were determined to be that of a sheep’s liver, as the lab demonstrators disclosed this information on the completion of gel electrophoresis. Upon reflection, it was concluded that despite the success of the experiment, there were certain aspects that were well monitored to ensure the attainment of results, making it difficult to specify any errors that could’ve been made if students completed these labs individually. For example, the DNA that students extracted was not used in the final experiment. Instead, lab demonstrators swapped this DNA over to one that was pre-filtered, purified, and diluted to utilise in the PCR process. In addition to this, students were also assisted with the gel electrophoresis process as the chamber, allele ladders and positive controls were already set up. By participating in this laboratory, it is clear that DNA is an incredible structure within any organism’s makeup, and because of this has proven extremely useful in research within scientific studies.
Bibliography
- Booth, C. S. et al., 2010. Efficiency of the polymerase chain reaction. Chemical Engineering Science, 65(17 ), pp. 4996–5006, doi: 10.1016/j.ces.2010.
- Brandt, C. G. & Gonzales, R. A., 2005. DNA Testing in Animal Forensics. Journal of Wildlife Management, 69(4), pp. 1454-1462 doi: 10.2193/0022-541X(2005)69[1454:DTIAF]2.0.CO;2.
- Ciampolini, R., Leveziel, H., Mazzanti,E., Grohs,C & Cianci, D. 2000. Genomic identification of the breed of an individual or its tissue. Meat Science, 54(1), pp. 35-40 doi: 10.1016/S0309-1740(99)00061-3.
- Cocciolone, R. A. & Timms, P., 1992. DNA Profiling of Queensland Koalas reveals Sufficient Variability for Individual Identification and Parentage Determination. Wildlife Research , 19(3), pp. 279-287.
- Lesk, A. M., 2005. Introduction to Bioinformatics. Second ed. Oxford: Oxford University Press.
- O'Sullivan, G., Sharman, E. & Short, S., 1999. The molecular biology explosion and Social Context. In: Goodbye Normal Gene. NSW: Pluto Press Australia Limited, pp. 14-16.
- Reed, R., Holmes, D., Weyers, J. & Jones, A., 2007. Molecular genetics II - PCR and related aplications. In: Practical Skills in Biomolecular Sciences. Essex: Pearson Education Limited, pp. 439-455.
- Sanderson, C. J., 2007. DNA: The template. In: Understanding Genes and GMOs. USA: World Scientific Publishing Co, pp. 6-20.
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Identifying An Unknown Tissue Sample Via DNA Extraction.
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