Applying UV-Vis for Successful Downstream GMO Detection

The controversy of grocery products containing genetically modified organisms (GMO) has heightened the demand for analytical testing in the food and beverage industry.^1,2 GMOs, or transgenic crops, contain DNA that was modified through bioengineering to introduce a desired trait that is not naturally occurring in the species.¹ Current legislation for regulating GMO-based products varies between countries, indicating the need for worldwide, reliable testing procedures.²

Qualitative PCR is usually implemented first to screen for the presence or absence of common regulatory elements used in transgenic (GMO) crops. Quantitative PCR is then performed, detecting more specific targets and providing a quantitative analysis on the target copy number.⁴ Completing a qualitative assessment prior to quantitative PCR can save time and resources by eliminating targets that were defined as absent.

There are two popular methods for producing transgenic crops with the designated DNA construct: particle bombardment and transformation via Agrobacterium.¹ With the particle bombardment method, the DNA construct is bound to particles which are accelerated at high velocity into plant tissues or cells to the nucleus. The DNA construct releases from the particles and integrates into the plant DNA via homologous recombination.^10,11 After the bombardment, the cells are transferred to a selective media based on the chosen selection agent in the DNA construct, and the plant regenerates until it is a fully grown transgenic plant.^10,12

With the Agrobacterium method, Agrobacterium naturally infects plants and uses its own tumor-inducing (Ti) plasmid to integrate transfer DNA (T-DNA) into the plant genome.¹⁰ When producing transgenic plants, the desired trait is inserted into the Ti plasmid, reintroduced to the Agrobacterium, and infects the plant. T-DNA containing the transgene integrates into the plant DNA, and cells are selected and regenerated as described with the particle bombardment method.^10,12

Globally, soybeans stand as the most popular transgenic crop, where herbicide tolerance is the most desired trait, and pest resistance follows close behind.³ In this study, soy DNA was isolated from commercial tofu, soy milk, edamame, and soybeans with a modified protocol from Doyle and Doyle (1987) using the ionic detergent cetyltrimethylammonium bromide (CTAB). The extracted DNA was measured via spectrophotometry to verify concentration and purity prior to qPCR, where the DNA was tested for the presence or absence of GMO targets via qPCR.

GMO detection has become simpler through multiplex qPCR assays where the targets are P35S/CaMV, TNOS/A. tumefaciens, and P34S/FMV. Because these regulatory elements are naturally found in their corresponding virus or bacteria, it is common to experience false GMO-positive reactions if the plant has been naturally infected.⁸ Multiplex qPCR allows simultaneous detection of the regulatory elements and the virus or bacteria to eliminate false-positive results. PCR is a sensitive assay that requires template DNA to be a specific amount and optimal purity to ensure a successful and reproducible reaction.¹³

To determine the quantity and quality of template DNA and ensure a successful qPCR run, a UV-Vis spectrophotometer was used to calculate concentration and identify contaminants from nucleic extractions. When extracting nucleic acids from food sources, proteins are abundant and frequently co-extracted if proper extraction technique is not followed. Contaminating proteins are known to inhibit the DNA polymerase in PCR, reducing PCR efficiency.¹⁴ Identifying common extraction contaminants, such as proteins and phenol, allows scientists to make simple modifications to extraction protocols without the need for extensive troubleshooting when PCR efficiency is poor.

When utilizing qPCR for GMO detection, data reliability is a top priority as food and beverage products must comply with government regulations on transgenic crops. Microvolume spectrophotometers are capable of quickly quantifying DNA, RNA, and protein from only 1–2 µL of sample to bring you more knowledge about your sample, so that you can save days of troubleshooting and accelerate your research, and help your lab stay compliant with federal data regulations.

You can read the results and detailed experimental procedures in this application note, Quantifying soy DNA extracts for downstream GMO detection.

References and Additional Resources

• Spectrophotometer Resources
• Application Note: Quantifying soy DNA extracts for downstream GMO detection
• References Note:  These references are gathered from the above article as well as those included in the application note referred to in the article:

1. Rani, S.J., & Usha, R. (2013). Transgenic plants: Types, benefits, public concerns and future. Journal of Pharmacy Research, 6, 879-883.
2. Turnbull C., Lillemo M., and Hvoslef-Eide TAK. (2021). Global Regulation of Genetically Modified Crops Amid the Gene Edited Crop Boom – A Review. Front. Plant Sci. 12:630396. doi: 10.3389/fpls.2021.630396.
3. James, Clive. (2011). Global Status of Commercialized Biotech/GM Crops: 2011. ISAAA Brief No. 43. ISAAA: Ithaca, NY.
4. Fraiture, M. A., Herman, P., Taverniers, I., De Loose, M., Deforce, D., & Roosens, N. H. (2015). Current and new approaches in GMO detection: challenges and solutions. BioMed research international, 2015, 392872. https://doi. org/10.1155/2015/392872.
5. Amack, S. C & Antunes, M. S. (2020). CaMV35S promoter – A plant biology and biotechnology workhorse in the era of synthetic biology. Current plant biology, 24, 100179. doi: 10.1016/j.cpb.2020.100179.
6. Holden, M. J., Levine, M., Scholdberg, T., Haynes, R. J., & Jenkins, G. R. (2010). The use of 35S and Tnos expression elements in the measurement of genetically engineered plant materials. Analytical and bioanalytical chemistry, 396(6), 2175–2187. https://doi.org/10.1007/s00216-009-3186-x.
7. Sanger, M., Daubert, S., & Goodman, R. M. (1990). Characteristics of a strong promoter from figwort mosaic virus: comparison with the analogous 35S promoter from cauliflower mosaic virus and the regulated mannopine synthase promoter. Plant molecular biology, 14(3), 433–443. https://doi.org/10.1007/BF00028779.
8. Bak A. and Emerson JB. (2020). Cauliflower mosaic virus (CaMV) Biology, Management, and Relevance to GM Plant Detection for Sustainable Organic Agriculture. Front. Sustain. Food Syst. 4:21. doi: 10.3389/fsufs.2020.00021.
9. Kim, K. J., Kim, H. E., Lee, K. H., Han, W., Yi, M. J., Jeong, J., & Oh, B. H. (2004). Two-promoter vector is highly efficient for overproduction of protein complexes. Protein science: a publication of the Protein Society, 13(6), 1698–1703. https://doi.org/10.1110/ps.04644504.
10. Homrich, M. S., Wiebke-Strohm, B., Weber, R. L., & Bodanese-Zanettini, M. H. (2012). Soybean genetic transformation: A valuable tool for the functional study of genes and the production of agronomically improved plants. Genetics and molecular biology, 35(4 (suppl)), 998–1010. https://doi.org/10.1590/s1415-47572012000600015.
11. . Sanford, J.C. (1990), Biolistic plant transformation. Physiologia Plantarum, 79: 206-209. https://doi.org/10.1111/j.1399-3054.1990.tb05888.x.
12. Yan, Y., Zhu, X., Yu, Y., Li, C., Zhang, Z., Wang, F., Nanotechnology Strategies for Plant Genetic Engineering. Adv. Mater. 2022, 34, 2106945. https://doi.org/10.1002/adma.202106945.
13. Lorenz T. C. (2012). Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. Journal of visualized experiments: JoVE, (63), e3998. https://doi.org/10.3791/3998.
14. Rossen, L., Nørskov, P., Holmstrøm, K., & Rasmussen, O. F. (1992). Inhibition of PCR by components of food samples, microbial diagnostic assays and DNAextraction solutions. International journal of food microbiology, 17(1), 37–45. https://doi.org/10.1016/0168-1605(92)90017-w.
15. Doyle, J.J. and Doyle, J.L. (1987). A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochemical Bulletin, 19, 11-15.