Lab 10: Exploring GMOs

Lab Objectives

• To understand how genetic engineering supplements traditional methods of plant breeding to generate new traits in crop plants

• To understand how changing the genome of an organism can affect its ability to survive in different environments

Notebook

Procedure Overview

Part 1: Extraction of DNA from food samples

Part 2: Set up PCR reactions

Part 3: Electrophoresis of PCR products

Part 4: Analysis of results

Notebook

Background Information

• First genetically modified (GM) crop was released in the US in 1994

• GM crops are controversial due to potential health and environmental risks

• Foreign genetic material from another plant or from other species

• Inserted gene codes for a protein that gives the plant an advantage over similar crop plants

Why genetically modify?

• Pest resistance

• Herbicide tolerance

• Delayed fruit ripening

• Improved fruit yield

• Increased nutrient content

GM Crops • Arctic Apple – resists browning

• Golden Rice – biosynthesize beta carotene

• Bt Corn – produces an endotoxin that is lethal to the European corn borer

• Vistive Gold – Round Up resistant soy bean

• Papaya – Resistant to papaya ring spot virus

How do you genetically modify a crop?

• Identify a trait (protein) that has the potential to improve a crop

• Isolate (clone) the gene that codes for the protein

• Engineer the gene so that the crop plant’s cells will read it correctly and manufacture the protein of interest

• Introduce the engineered gene into the plant

• Backcross the GM crop into the highest-yielding crop in the field

GMO-Associated Sequences

• 35S promoter of the cauliflower mosaic virus (CaMV 35S)

• Terminator of the nopaline synthase (NOS) gene of Agrobacterium tumefaciens

• Both sequences are present in ~85% of GM crops around the world

For or Against GMOs?

• Reduce use of herbicides and pesticides

• Preserve land

• Improve nutritional value of food

• Drought tolerance

• Salinity tolerance

• Longer shelf life

• Potential for “superweeds” or “superbugs”

• Possible allergens

• Not enough research

• Unpredictable changes to an ecosystem

• Antibiotic resistance

Recommended Foods to Test • Processed food such as cheese-flavored puffed

corn snacks (GM corn)

• Inexpensive meat products that contain soy fillers (GM soy)

• Nonorganic foods

• Papaya products

• AVOID fresh corn or soy

GM Food Labeling Legislation

• United States: Food is labeled “GM Free” if the food is < 5% GM

• European Union: Food is labeled “GM” if the food is > 1% GM

• Japan: Food is labeled “GM” if the food is > 5% GM

Test Foods Notebook

Part 1: Extraction of DNA from Food Samples

• Two microcentrifuge tubes – label as follows:

–Non-GMO (with group initials)

– Test Food (with group initials)

• Prepare 50 µL of Non-GMO ground slurry and 50 µL of Test Food ground slurry

• Prepare the non-GMO sample first to avoid contamination!

Notebook

Part 2: PCR Workstation

• Ice bath

• GMM: GMO Master Mix with primer (ice)

• PMM: Plant Master Mix with primer (ice)

• GMO +: GMO Positive Control DNA (ice)

• Test Food DNA

• Non-GMO Food Control DNA (oats)

• 6 PCR tubes and 6 PCR Adaptors

• 2-20 µL micropipette with tips

• Plant primers (PMM) – Used to determine if DNA was extracted from the plant material

– Targets a 455 bp region of a chloroplast gene

• GMO primers (GMM) – Used to determine if the food contains GMOs

– Targets a 203 bp fragment of the CAMV 35S promoter and a 225 bp fragment of the NOS terminator

Part 2: Set Up PCR Reactions

Notebook

Part 2: Set Up PCR Reactions

• Number the tubes #1-6

• Label clearly with group initials

Notebook

Purpose of Each PCR Reaction

Notebook

Part 3: Gel Electrophoresis of PCR Products

• First, add 10 µL of Orange G loading dye to each PCR sample (use a fresh tip each time) – gently tap the tubes to mix

• Load 20 µL of each sample into the wells according to the chart provided

• Load 20 µL of the molecular weight ruler (MWR) into Lane 7

Notebook

Part 3: Gel Electrophoresis of PCR Products

• Use a 3 % gel for electrophoresis of PCR products

• Run at 100 volts for 30-45 minutes

• Record a picture of your gel using UV light

Notebook

Part 4: Analysis of Results

• Draw the results of gel electrophoresis into your lab notebook

• Be sure to label each well on the gel picture

• Label the base pair size for each fragment of the ladder (molecular weight ruler)

• Write a conclusion based on the results of your test food

• Discuss any sources of error that may have affected your results

Notebook

Analysis of Results