Quantitative and qualitative analysis of three DNA extraction methods from soybean, maize, and canola oils and investigation of the presence of genetically modified organisms (GMOs)

Abstract

The objective of this study was to develop a DNA-based method for the identification and tracking of edible oils, which is important for health management. Three different DNA extraction methods (CTAB, MBST kit, and manual hexane-based method) were used to obtain high-purity DNA from crude and refined soybean, maize, and canola oils. PCR was then conducted using specific primers to identify the presence of genes related to each oil type and to assess transgenicity. The results showed that DNA was present in crude and refined oils, but in very low amounts. However, using method 3 for DNA extraction provided sufficient quantity and quality of DNA for successful PCR amplification. The study concluded that the main challenge in DNA extraction from oils is the presence of PCR inhibitors, which can be overcome using the manual hexane-based method. Also, the examination of protein presence in the oils using SDS-PAGE did not indicate any protein bands.

Keywords: Acetic acid glacial (PubChem CID: 176); CTAB (PubChem CID: 5974); Chloroform (PubChem CID: 6212); Coomassie brilliant blue G-250 (PubChem CID: 6324599); Crude and refined edible oils; DNA extraction; Ethanol (PubChem CID: 702); Genetically modified organisms (GMOs); Hexane (PubChem CID: 8058); Isopropanol (PubChem CID: 3776); Methanol (PubChem CID: 887); PCR inhibitors; Polymerase chain reaction (PCR).

Fig. 1

Agarose gel electrophoresis of genomic DNA extracted from crude Soybean Oil. Well 1: DNA extraction using the CTAB method, wells 2 and 3: DNA extracted using the MBST kit, well 4: DNA extracted using the manual hexane-based method, M: Molecular marker.

Fig. 2

Agarose gel electrophoresis of PCR products amplified from DNA extracted from crude and refined soybean oil using the manual hexane-based method. A: Primary PCR, B: Secondary PCR, C⁻: Negative control (water), C⁺: Positive control (soybean leaf DNA), well 1: pellet phase crude soybean, well 2: liquid phase crude soybean, Well 3: pellet phase refined soybean, well 4: liquid phase refined soybean, wells 5 and 6: crude and refined soybean oil were directly added as a template to the PCR reaction, respectively, M: Molecular marker (bp 100).

Fig. 3

Agarose gel electrophoresis of PCR products amplified from DNA extracted from crude and refined corn oil using the manual hexane-based method. C⁻: Negative control (water), C⁺: Positive control (corn leaf DNA), wells 1 to 4: 0.5, 1.0, 1.5, and 2.0 µl of DNA extracted from pellet phase crude corn as templates in PCR reaction, respectively, well 5: pellet phase refined corn, well 6: liquid phase refined corn, well 7: refined corn oil were directly added as a template to the PCR reaction, M: Molecular marker (100 bp).

Fig. 4

Agarose gel electrophoresis of PCR products amplified from DNA extracted from crude and refined canola oil using the manual hexane-based method. C⁻: Negative control (water), C⁺: Positive control (canola leaf DNA), well 1: pellet phase crude canola, well 2: pellet phase refined canola M: Molecular marker (100 bp).

Fig. 5

Agarose gel electrophoresis of PCR products amplified from DNA extracted from crude and refined soybean, corn, and canola oils using NOS primers. C⁻: Negative control (water), C⁺: Positive control (plasmid containing NOS sequence), well 1: pellet phase crude soybean, well 2: liquid phase crude soybean, wells 3: pellet phase refined soybean, well 4: liquid phase refined soybean, well 5: pellet phase crude corn, well 6: pellet phase refined corn, well 7: pellet phase crude canola, well 8: pellet phase refined canola, M: Molecular marker.

Fig. 6

Quality assessment of extracted protein from oils. Well 1: protein extracted from leaf, well 2: refined Soybean, well 3: refined Canola, well 4: refined Corn, well 5: Crude Soybean, well 6: Crude Canola, well 7: Crude Corn, M: Molecular weight marker.