GMO Practical Study Notes

BY450 Fundamentals of Genetics & Evolution GMO Practical

Introduction to GMO Crops

  • Background: Since the first genetically modified (GM) crop was released in the US in 1996, the use of these crops has been debated due to potential health and environmental risks.

  • Testing and Labeling: GM foods must be labeled if they contain more than 1% GM content in Europe and Asia.

GMO Investigator Kit Overview

  • The GMO Investigator kit utilizes PCR (Polymerase Chain Reaction) to detect GM genomes.

  • Key Sequences Detected:

    • 35S promoter: Associated with the cauliflower mosaic virus (CaMV 35S).

    • NOS terminator: Derived from the nopaline synthase gene of Agrobacterium tumefaciens.

  • Presence of Sequences: One or both sequences are found in most GM crops.

  • Components: Promotor, Ti plasmid, transgene, terminator, and gene cassette.

PCR Outcomes

  • Outcomes of PCR:

    • Successful PCR: Known as a positive outcome (+ve outcome).

    • Unsuccessful PCR: Known as a negative outcome (-ve outcome).

    • True Outcomes:

    • True +ve: Genuine positive result.

    • False +ve: Contamination leads to a false positive.

    • True -ve: Genuine negative result (no plant DNA).

    • False -ve: Contamination or damaged DNA leads to a false negative.

Practical Procedures

Setting Up PCR Reactions

  1. Integrity of Plant DNA:

    • Test for photosystem II chloroplast gene using PCR.

    • A +ve outcome indicates the presence of plant DNA; a -ve outcome indicates no plant DNA is present.

  2. Testing PCR Integrity:

    • Amplify 35S promoter and photosystem II gene sequences from control DNA to ensure PCR is functioning.

    • A +ve outcome indicates functioning PCR; a -ve outcome indicates PCR failure.

  3. Contamination Testing:

    • Using DNA from a certified non-GMO food control to identify contamination of test samples.

    • A +ve result indicates contamination; a -ve result indicates no contamination detected.

DNA Extraction Procedure

  1. Extract DNA from Food Samples:

    • Choose a food sample (weigh 0.5-2g).

    • Label screwcap tubes:

      • One for a non-GMO control.

      • One for the test sample.

    • Add 500 µL of InstaGene to each tube.

    • Add 5 mL of distilled water for every gram of food by calculating water volume:
      ext{Mass of food (g)} imes 5 = ext{Volume of water (mL)}

    • Grind food for 2 minutes until a slurry is formed; repeat grinding with additional water until smooth.

    • Pipette 50 µL of slurry into the respective screwcap tubes and vortex to mix.

    • Place in a 95°C water bath for 5 minutes and then centrifuge for 5 minutes.

Setting Up PCR Reactions

  1. Number and Identify PCR Tubes:

    • Number small PCR tubes from 1 to 6 and mark for identification.

    • Corresponding tube contents:

      • Tube 1: Plant MM (photosystem II primers) + Non-GMO control DNA

      • Tube 2: GMO MM (35S promoter primers) + Non-GMO control DNA

      • Tube 3: Plant MM + Test food DNA

      • Tube 4: GMO MM + Test food DNA

      • Tube 5: Plant MM + GMO positive control DNA

      • Tube 6: GMO MM + GMO positive control DNA

  2. Adding Master Mix and DNA to Tubes:

    • Add 20 µL of master mix to each PCR tube using a fresh tip for each addition.

    • Avoid placing tips into the InstaGene pellet at the bottom during pipetting.

    • Place on ice until ready for the thermal cycler.

Thermal Cycling Parameters

  • Thermal Cycling Steps:

    • Initial Denaturation: 94°C for 2 minutes

    • PCR Cycles: 40 cycles featuring:

    • Denaturation: 94°C for 1 minute

    • Annealing: 59°C for 1 minute

    • Extension: 72°C for 2 minutes

    • Final Extension: 72°C for 10 minutes

    • Hold: 4°C indefinitely

Linkage and Recombination Discussed in Class

Genetic Recombination

  • Haplotypes: Shuffling of haplotypes through genetic recombination during meiosis.

  • Chromatid Alignment: When chromosomes align, chromatids can crossover, leading to formation of new haplotypes.

  • Linkage Disequilibrium: Haplotype combinations can become linked if genes are physically close on a chromosome. The ratio of recombinant (R) and non-recombinant (NR) gametes informs about gene proximity on the chromosome.

Examples of Gene Frequencies

  • Population Example:

    • Gene #1: alleles G and C; frequencies: pG = pC = 0.5

    • Gene #2: alleles A and T; frequencies: pA = pT = 0.5

    • Frequencies calculated as follows:

      • ext{Frequency of G-A} = rac{2}{8} = 0.25

      • ext{Frequency of G-T} = rac{2}{8} = 0.25

      • ext{Frequency of C-A} = rac{2}{8} = 0.25

      • ext{Frequency of C-T} = rac{2}{8} = 0.25

  • Findings indicate unlinked alleles behaving randomly.

  • In contrast, if certain combinations appear frequently, genes are suggested to be linked, indicating strong linkage disequilibrium.

Conclusion

  • Through the practical, students test food and animal feed samples for the presence of GM genomes, gaining insights into molecular genetic techniques and the implications of genetic engineering in agriculture.