Introduction
General Purpose of GMOs
There are various reasons why a plant’s gene is altered through scientific and artificial means. Improving crop yields is one of the primary purposes of GMOs, as genes for rapid growth are introduced (Kavhiza et al., 2022). Additionally, the alteration can be used to increase pest and disease resistance (Kavhiza et al., 2022). Unlike natural genes, modified genes have greater resistance to pests and diseases. Furthermore, crops can be modified to increase and improve their nutritional value.
Production and Examples of GMOs
The scientists first identify a desired trait that can be transferred to a naturally growing plant. After that, the desired trait is cloned into a delivery vector (Steffen et al., 2022). The carrier is genetically engineered to carry the cloned information. The transgene is then delivered into plant cells with undesired traits to improve them. The last step involves selecting plant cells with the new traits and cultivating them. Potatoes, soya beans, sugar beets, summer squash, and apples are examples of foods whose genetic components are modified.
Deoxyribonucleic Acid (DNA) Extraction
DNA extraction involves isolating molecules that carry genetic information from cells. Cell lysis is the initial step of digestion, during which cells are disintegrated, releasing their contents, including molecules (Steffen et al., 2022). Organic solvents are often used for protein denaturation, altering the structure of the cell lysate.
Methods such as column chromatography are used to remove impurities. Heat is used to denature proteins and lyse cells. Metal ions that may interfere with the polymerase chain reaction (PCR). Therefore, Chelex 100 is used to remove metal ions.
PCR
PCR works by continuously copying a target sequence using an enzyme called Taq DNA polymerase. The latter enzyme works best in high temperatures by adding deoxynucleotide triphosphate to the end of the growing DNA strand (Jabbour, n.d.). Thermocyclers are used for PCR, allowing denaturation, annealing, and extension of DNA polymerase copies. Therefore, Taq DNA is heat-resistant and plays a vital role in PCR.
Gel Electrophoresis
Gel electrophoresis is a laboratory method used to separate DNA based on molecular size. Agarose gel electrophoresis uses a loading buffer that contains a tracking dye, which serves to visualize, act as a density agent for sinking, and alter the secondary structures of molecules. The electrophoresis buffer solution performs two functions: enhancing ion conduction and maintaining pH (Jabbour, n.d.).
The property of DNA that enables gel electrophoresis is its negative charge, arising from the availability of phosphate groups. Therefore, they move towards a positively charged electrode, allowing their separation. DNA fragments move at different rates due to their varying molecular sizes, with the larger ones moving slowly.
Overall Purpose of the Lab Experiment
The experiment was conducted to test for the presence of GMO genes in papaya food samples. Three other food samples were included in the experiment for positive and negative control. Identifying the presence of GMO genes is essential for understanding a plant’s traits and characteristics.
Methodology
Various steps were followed to identify GMOs in papaya successfully. Heat was applied to the food sample to denature and lyse papaya cells. A mortar-and-pestle homogenizer was used to grind the papaya sample. After that, Chelex 100 was added to remove metal ions that could interfere with the PCR. The initial steps helped in extracting DNA from the papaya before it was subjected to further analysis.
PCR was done to amplify the DNA sequence in the prepared papaya sample. Master mixes containing plant and GMO primers were prepared. The primers targeted particular regions in the papaya sample. The procedure for programming the thermocycler was provided in the lab manual (Jabbour, n.d.). According to the manual, denaturation was to be performed by heating the tube containing the food sample to 94 °C. Annealing and extension were done at 55 °C and 60 °C, respectively (Jabbour, n.d.). The PCR made the sample read for gel electrophoresis. The process was repeated 30 times to achieve maximum DNA amplification.
The final step involved gel electrophoresis, which separated the DNA fragments based on their molecular sizes. SYBR® Green is the dye that was used as a nucleic stain during gel electrophoresis. The dye fluorescently stained the DNA bands, making them easily distinguishable and allowing the presence of GMOs to be observed. The process was repeated for three different food samples, and the data were recorded in the lab worksheet. Figure 1.0 shows the three gel electrophoreses.
Analysis
The experiment involved four food samples, including papaya, and three unidentified samples. Both samples were subjected to three different PCRs: an actual experiment, a negative control, and a positive control. Gel 1 was the positive control, and Gel 3 was the negative control. Meanwhile, gel number 2 was the actual experiment, which was compared to the other two. The size of the PCR was compared to that of the ladder.
All the food samples were negative for GMO genes, but some showed amplified DNA. Compared to papaya, the second food showed its bands visible farther from the negative electrode. Additionally, the third food was nearer to the positive electrode than the papaya food sample. Thus, the food molecules are smaller than those of papaya. The fourth sample tested negative with no bands visible, indicating unsuccessful amplification. Therefore, the presence of bands indicated successful DNA amplification, while the absence of bands indicated unsuccessful amplification.
There are various sources of error for the sample. Inconsistent sample preparation and contamination could be the causes of the error. Additionally, incorrect primer design may result in inefficient binding of target molecules. Furthermore, inhibitors in the nucleic acid extract may interfere with PCR.
Conclusion
General Summary
The lab experiment involved extracting DNA from papaya and three other food samples. To identify the presence of modified genes in papaya, DNA was isolated from the plant samples. After that, the master mixers with primers were subjected to PCR to identify GMO genes.
Data Summary
The experiment was conducted to identify the presence of modified genes in papaya. Although all the papaya food samples tested negative for GMO genes, amplified DNA was detected. However, the second and third food samples were more amplified than papaya. The fourth sample showed no amplification, which could be due to various errors.
Sources of Error and Suggestions
Inhibitors, inconsistent food sample preparation, and incorrect primer design could be the causes of errors during the experiment. The fourth food sample was the most affected, but that can be improved in future experiments. Strict adherence to the lab experiment manual and proper primer design could help prevent future errors.
Lessons Learned
Genetically modified crops are more resistant to diseases and pests than non-genetically modified crops. The process of creating GMO genes involves identifying the desired trait, cloning the gene, and delivering it to the target organism. After extracting DNA from a plant, PCR can be used to detect GMO genes.
References
Jabbour, M. (n.d.). Laboratory manual for general biology II. GCU.
Kavhiza, N. J., Zargar, M., Prikhodko, S. I., Pakina, E. N., Murtazova, K. M.-S., & Nakhaev, M. R. (2022). Improving crop productivity and ensuring food security through the adoption of genetically modified crops in sub-SaharanAfrica. Agronomy, 12(2), 439.
Steffen, C. R., Romsos, E. L., Kiesler, K. M., Borsuk, L. A., Gettings, K. B., & Vallone, P. M. (2022). Make it “SNPPY” – updates to SRM 2391d: PCR-based DNA profiling standard. Forensic Science International: Genetics Supplement Series, 8, 9–11.