Activity 3-1 - Identifying Genetically Modified Organisms Through DNA Analysis

GMO Detection Guided Approach

The GMO detection kit used in this activity encourages a guided inquiry-based learning method. This means students follow structured protocols but also make critical decisions along the way—like choosing appropriate controls, troubleshooting PCR issues, and interpreting gel results. Students will ask questions like:

• Was DNA extraction successful? A control PCR reaction targeting a plant gene (e.g., tubulin or rbcL) ensures the sample contains amplifiable plant DNA.
• Did PCR work as expected? Positive and negative controls help interpret results. A successful PCR will show clear bands on a gel, while no bands could suggest failed amplification.
• Is there contamination? Contamination can lead to false positives. Students will use sterile technique and proper controls to validate their results.

GM Crops

The experiment doesn’t end at the gel. Students are encouraged to explore the broader implications of GMO technology by engaging in a structured debate on its risks and benefits.

Concerns About GM Crops:

• Super-Weeds: When herbicide-resistant crops like “Roundup Ready” soybeans cross-pollinate with wild relatives, the result can be herbicide-resistant weeds that are harder to control.
• Super-Bugs: Insects exposed to Bt crops over many generations may evolve resistance, rendering the Bt trait ineffective.
• Allergic Reactions: Introducing new genes can lead to the expression of novel proteins. If these proteins resemble allergens, they could pose a health risk to sensitive individuals.
• Antibiotic Resistance: Some GM crops use antibiotic resistance genes as markers during development. There is concern these genes could be transferred to gut bacteria, potentially contributing to antibiotic resistance.
• Public Health Risks: While most GMOs are considered safe, critics argue that long-term health effects have not been fully studied, particularly for traits affecting metabolism or protein expression.

Benefits of GM Crops:

• Environmental Impact: GM crops that require fewer chemical pesticides reduce environmental pollution and protect beneficial insect populations.
• Soil Conservation: Crops engineered for drought resistance or pest resistance can grow in less-than-ideal soil, reducing the need to clear additional farmland.
• Nutritional Benefits: “Golden rice,” genetically engineered to produce beta-carotene, helps combat vitamin A deficiency in regions where rice is a staple food.
• Agricultural Expansion: GM technology enables farming in areas previously unsuitable for agriculture due to poor soil or harsh climates, improving food security.

Through this lab, students develop a comprehensive understanding of how molecular biology can be applied to everyday problems—like verifying the presence of GMOs in food. They not only learn lab techniques like DNA extraction, PCR, and gel electrophoresis, but also explore the real-world implications of genetic engineering. By combining technical skills with ethical and environmental discussions, this activity fosters critical thinking and gives students the tools to make informed decisions about biotechnology and its role in our global food system.

Objective

Test store-bought plant-based foods (like corn chips or veggie burgers) to check for the presence of genetically modified organisms (GMOs) using DNA extraction, PCR, and gel electrophoresis.

Diluting Primers (Stock Prep)

• For every 1 nmol of dried primer, add 10 µL of distilled water (diH₂O).
  • → This creates a 100 µM stock solution (also called 100X).

Make a Working Primer Stock (for each primer set)

• Label a new microcentrifuge tube with: "Working Stock" – Gene Name – Group Name
• Mix:
  • 240 µL diH₂O
  • 5 µL Forward primer
  • 5 µL Reverse primer
    • → This gives a 2 µM working solution (10X primer mix)

Primer Selection

To conserve our limited reagents, you'll be working in pairs instead of individually. There are five different GMO-related primer sets available (plus one control). Each pair can choose up to three primers to test. Please select your primers from the list below and explain what each one detects in your lab notebook or worksheet.

⚠️ Important Note:

• If your sample tests positive for any of these GMO markers, that means the food contains a GMO sequence.
• However, if your result is negative, that does not mean the food is organic — it may still contain GMOs that we aren’t testing for.
  • We are only using a limited number of primers, so a negative result simply means that none of the specific sequences tested were found.

Primer Options

1. (REQUIRED) Plant Master Mix (Control)
   • Purpose: This is not a GMO marker. It’s a positive control to confirm your DNA extraction worked and that plant DNA is present in your sample.
   • Why it’s used: If this doesn't show a band in PCR, it means your extraction or PCR failed — not that the food is GMO-free.
2. Invertase
   • Purpose: Detects the invertase gene, which helps plants break down sucrose into glucose and fructose.
3. Sucrose Phosphate Synthase (SPS)
   • Purpose: SPS is involved in the biosynthesis of sucrose, an important sugar in plants.
4. Lectin
   • Purpose: Detects the lectin gene, a natural antimicrobial gene.
5. 35S Promoter
   • Purpose: This sequence comes from the Cauliflower Mosaic Virus and is used to turn on genes in GMO crops.
   • Why it's engineered: It acts like an "on-switch" to make sure inserted GMO genes are actively expressed in the plant. It’s one of the most common elements in GMO crops.
6. NOS Terminator
   • Purpose: This sequence comes from the nopaline synthase gene in Agrobacterium tumefaciens.
   • Why it's engineered: It acts like a "stop sign" for transcription, making sure the inserted GMO gene turns off properly after it's expressed. It’s often paired with the 35S promoter in GMO constructs.

Materials:

• Food sample (one sample per pair), distilled water, grinder, microcentrifuge tubes

Procedures

1. Weigh 1 gram of your plant-based food sample.
2. Add 5 mL of distilled water.
3. Grind or mash the sample for 2 minutes until well mixed.
4. Divide your food slurry into 3 tubes:
   • 50 µL into a tube labeled “For Gel”
   • 500 µL into a tube labeled “RT-PCR” → Freeze
   • 500 µL into a tube labeled “Sequencing” → Freeze
   • Freeze down the remaining solution.
5. Add 500 µL of InstaGene solution to your “For Gel” tube.
6. Boil this sample for 5 minutes via heat block at 95oC
7. Spin the tube in a microcentrifuge for 10 minutes.
8. Carefully transfer the clear supernatant to a new tube and label it “For Gel”
   • You can discard the original tube with the pellet.
9. Use the Nanodrop to measure your DNA concentration.
10. Record the concentration: ______ ng/µL
    • If your sample is more concentrated than 100 ng/µL, dilute it:
      • Add diH₂O to bring the concentration down to 100 ng/µL.
    • If the concentration is already less than 100 ng/µL, no dilution is needed.
11. Calculate how much DNA you'll need for your PCR:
    • You need 200 ng total DNA per reaction.
    • Example: If your DNA is 50 ng/µL → 200ng ÷ 50ng/µL = 4 µL needed
12. Add enough water to bring the total to 12 µL
    • If 4 µL DNA, add 8 µL water.

Materials:

• Agarose, TAE buffer, SYBR dye, gel box, loading dye, UV transilluminator

Procedures:

1. Prepare a 0.8% agarose gel:
   • Mix 0.24 g of agarose into 30 mL of 1X TAE buffer.
   • Microwave until fully dissolved.
   • Let it cool (warm to the touch), then add:
   • 3 µL of 10,000X SYBR Safe dye (for DNA visualization)
   • Pour the gel into a casting tray and let it set in the dark.
2. Mix your samples before loading:
   • 20 µL of your PCR sample
   • 4 µL of 6X DNA loading dye
3. Carefully load your samples into the gel wells.
4. Run the gel in 1X TAE buffer for 30–45 minutes, in the dark.
5. Visualize your results under UV light or a blue light transilluminator.
6. Compare your bands to the GMO-positive and GMO-negative controls.