Greenomes – Plant Molecular Genetics and Genomics
Detecting Genetically Modified Food by PCR
Most Americans would probably be surprised to learn that more than 60% of fresh vegetables and processed foods sold in supermarkets today, have been altered by direct gene transfer. Over 150 million acres of farmland worldwide are used to grow genetically modified (GM) crops. The most widely planted GM crops are corn, soybeans, and cotton.
This laboratory uses a rapid method to isolate DNA from plant tissue and to genotype a soy plant using the polymerase chain reaction (PCR) method. The 35S promoter, which drives expression of the glyphosate resistance gene, is identified in soy plants that are resistant to the herbicide "Roundup”. Resistance correlates with an insertion allele that is readily separated from the wild-type allele by electrophoresis on an agarose mini-gel. The technique can then be extended to assay common foods for molecular evidence of genetic modification.
Objectives/Goals:
This laboratory provides practical insight into:
· the relationship between genes, proteins, and traits.
· three methods (DNA extraction, PCR, and gel electrophoresis) that are commonly used in biological research.
· gene transfer technology and its impact on society.
INTRODUCTION
Methods in genetic engineering are producing a revolution in agriculture. Genes that encode for herbicide resistance, insect resistance, draught tolerance, frost tolerance, and other traits have been added to numerous plants of commercial importance- including strawberries, soy, corn, potatoes, rice, wheat, and canola.
A common approach to transferring new genes into different plant species makes use of the pathogenic plant bacterium Agrobacterium tumefaciens. In the wild, infection by this bacterium causes plant tumors, called crown galls. These tumors provide nutrients for the bacteria’s successful colonization of the plant. Galls are produced by the expression of genes contained on Agrobacterium’s tumor inducing (Ti) plasmid. The Ti plasmid contains a region of oncogenic DNA, flanked by direct repeats, that is transferred to the host cell. This mobile region of the Ti plasmid, called T (transfer) DNA, inserts randomly into the plant cell genome. Once inserted into the plant genome, the infected cell begins to express genes located on the T-DNA. The host cell then expresses genes on the T DNA needed by the pathogen. By placing DNA of interest within a modified T DNA region, researchers are able to use Agrobacterium to introduce foreign DNA into plants.
Agrobacterium-mediated gene transfer involves the use of two plasmids and is referred to as a “binary system”. First, the gene of interest is cloned into a small plasmid. This plasmid contains the direct repeat regions of the Ti plasmid, which allows efficient transfer of DNA into the plant cell genome. A polylinker containing multiple recognition sites allows one to splice a gene of interest between the direct repeats. The gene is inserted adjacent to a strong promoter-such as the 35S promoter from cauliflower mosaic virus. The plasmid also contains a selectable marker, the kanamycin-resistance gene, which allows successfully transformed plants to be identified by antibiotic selection. After subcloning the gene of interest, the plasmid is purified from E. coli and inserted into a strain of Agrobacterium that contains the second plasmid of the binary system. This plasmid is a larger, modified form of Ti, which carries genes required for infection and T-DNA transfer.
The genetically modified Agrobacterium is then used to infect a host plant. In Arabidopsis, ovules can be transformed simply by dipping early stage flowers in a suspension of Agrobacterium. After fertilization, a small proportion of seeds will produce genetically modified offspring.
Agriculture has been greatly impacted by the advances in gene transfer methods. One example has been the introduction of a gene that provides resistance to the broad-spectrum herbicide glyphosate, commonly known as “Roundup”. Crops genetically altered to possess this gene are referred to as “Roundup Ready”. These crops are resistant to the herbicide, while the invasive weeds are not. The major advantages of the "Roundup Ready” system include better weed control, reduction of crop injury, higher yield, and lower environmental impact than traditional herbicide systems.
REFERENCES
Castle, L.A., Siehl, D.L., Gorton, R., Patten, P.A., Chen, Y.H., Bertain, S., Cho, H.J., Wong, N.D., Liu, D., Lassner, M.W. (2004). Discovery and Directed Evolution of a Glyphosate Tolerance Gene. Science 304: 1151-1154.
Edwards, K., Johnstone, C. and Thompson, C. (1991). A Simple and Rapid Method for the Preparation of Plant Genomic DNA for PCR Analysis. Nucleic Acids. Res.19: 1349.
Stalker, D.M., McBride, K.E., Maiyj, L.D. (1988). Herbicide Resistance in Transgenic Plants Expressing a Bacterial Detoxification Gene. Science 242: 419-423.
Vollenhofer, S., Burg, K., Schmidt, J., Kroath, H. (1999). Genetically Modified Organisms in Food Screening and Specific Detection by Polymerase Chain Reaction. J. Agric. Food Chem. 47: 5038-5043
REAGENTS, SUPPLIES, AND EQUIPMENT
Reagents / Supplies and EquipmentWild-type and Roundup Ready soybean seed
Assorted dry food products
Edward’s buffer, 4 mL
Isopropanol, 1.5 mL
Tris/EDTA (TE) buffer, 400 mL
35S1/35S2 primer/loading dye mix*, 75 mL
Tub5/Tub3 primer/loading dye mix*, 75 mL
Ready-To-GoTM PCR beads
Mineral oil, 5 mL (depending on thermal cycler)
Marker, pBR322/BstN1* (9.75 mg), 130mL
10X TBE, 300 mL
Agarose, 2 g
Ethidium bromide (1 mg/mL), 250 mL
or
CarolinaBLUä gel and buffer stain, 7 mL
CarolinaBLUä final stain, 250 mL
*Store in a -20°C freezer / Seed growing tray
Planting container
Potting soil
Pellet pestles
Permanent markers
1.5 mL microcentrifuge tubes
Micropipets and tips (for measuring volumes from 2.5 mL to 900 mL)
Microcentrifuge tube racks (or empty pipet tip boxes)
Microcentrifuge
Vortex (optional)
Ice buckets with crushed ice
Thermal cycler
Water bath or heating block for boiling samples
Gel electrophoresis chamber and power supply
Staining trays
Latex gloves
White light box (to visualize DNA with CarolinaBLU)
UV transilluminator (to visualize DNA with ethidium bromide)
LAB FLOW AND SUMMARY
Note: You must plant the soy seed 2-3 weeks prior to performinging the lab.This lab can be broken into three parts:
I. Isolating DNA from soy plant tissue and dry food products using Edward's buffer.
II. Amplifying the 35S promoter and tubulin locus by PCR. During this step, specific primers are used to analyze the wild-type and the transformed plants having the 35S promoter.
III. Analyzing the amplified DNA by agarose gel electrophoresis.
The following table will help you to plan and integrate the three parts of the experiment.
Part / Day / Time / ActivityPreparation of
soy plants / 2-3 weeks before lab / 15-30 min. / Plant soy seeds
I. DNA Isolation / 1
/ 30 min. / Pre-lab: Set up student stations
30-60 min. / Isolate soy DNA
II. PCR Amplification / 2 / 30-60 min. / Pre-lab: Set up student stations
15-30 min. / Set up PCR reactions
70+ min. / Post-lab: Amplify DNA in thermal cycler
III. Analyzing Amplified
DNA by Gel
Electrophoresis / 3 / 30 min. / Prepare agarose gel solution and cast gels
4 / 30 min / Load DNA samples into gels
30+ min. / Electrophorese samples
20+ min. / Post-lab: Stain gels
20 min. to
overnight / Post-lab: De-stain gels
20 min. / Post-lab: Photograph gels
GROWING SOY
The table below provides a list of items for growing soy plants. Most of the supplies are from Hummert but many of them can be substituted with products that are easily obtainable from a hardware store.
Item / Description / SupplierShelving / Any shelves can be used that can house fluorescent light fixtures so that lights are 12-18 inches from the plants. / Hardware store
Fluorescent light fixtures and bulbs / Light fixtures should hold at least two 40 watt fluorescent bulbs. The lights should be “daylight” lights as opposed to cool white lights. / Hardware store
Timer / A timer is needed if plants are to be maintained on a cycle of 16 hours of light and 8 hours of dark. Plants can also be grown in 24 hours of light if a timer is not available. / Hardware store
Soil / The potting soil to grow soy is Metro Mix 200 (Catalog # 10-0325). Fertilizer and insecticide can be added as required. / www.hummert.com
Trays / Soy plants are watered from above so planting pots should be placed in plastic tray with holes (Catalog # 11-3000-1). The industry standard for trays is a 10” X 20” plastic flat. The planting pot inserts fit directly into these trays. / www.hummert.com
Pots / The inserts for the trays commonly used contain 8 planting pots (Catalog # 11-0250-1). There are also other inserts that can be used if a different number of planting pots are required. / www.hummert.com
Planting Seeds
Obtain wild-type and Roundup Ready soy seed. Plant seeds as described below and allow for a 2-week growth period.
1. Place the planting pots into a plastic tray containing holes. The holes will allow excess water to drain from the soil when it is initially dampened. Fill the planting pots evenly with soil but do not pack the soil tightly. Note: For the best results, obtain or make a potting soil formulated specifically for growing soy seed.
2. Plant the seeds 0.5 inches below the soil using your finger. Leave 3 to 4 inches of space between them to allow optimal growth and to easily visualize the plant phenotype.
3. Place the planting container into the growing tray. Water the plants from above to prevent the soil from drying out. However, do not allow the soil to remain soggy.
4. Grow the plants close to a sunny window at room temperature or slightly warmer.
5. Harvest plant tissue for PCR as soon as the plant’s first leaves become visible. This should be about 2 weeks after planting – depending on light and temperature conditions.
PART I: ISOLATING DNA FROM SOY AND DRY FOOD PRODUCTS
Pre-lab Notes
Dry Food Products
Dry food products work best using the DNA extraction protocol outlined below. Food products should contain either soy or corn as an ingredient. Products that have been tested successfully using this procedure include Doritos brand tortilla chips, Tostitos blue corn chips, Betty Crocker Bacos, Jiffy corn muffin mix, Pepperidge Farm Sausalito cookies, Almased multi-protein powder, and Meow Mix cat food.
Pre-lab Set Up
Each station serving two students working as a team should have:
Wild-type and Roundup Ready soy plants
Dry food product
Edward's buffer, 4 mL
Isopropanol, 1.5 mL
Tris/EDTA (TE) buffer, 400 mL
6- 1.5 mL microcentrifuge tubes
Permanent marker
3- Disposable pellet pestles
20-200 mL micropipet and tips
100-1,000 mL micropipet and tips
Microcentrifuge
Water bath or heating block to boil samples
Vortex (optional)
Ice bucket with crushed ice
Procedure
1. Obtain one wild-type and one Roundup Ready soy plant. From each plant, take two pieces of leaf tissue approximately 1/4 inch in diameter. (The large end of a 1,000 mL pipet tip will punch disks of this size.) Place the leaf tissue in separate microcentrifuge tubes, and label with plant type and your group number.
2. For dry food products, obtain a 2-3 mm piece of food product and place in a separate microcentrifuge tube, and label with sample type and group number.
3. Add 100mL of Edward’s buffer to each tube containing the plant or food material. Grind the plant tissue or food product forcefully in the microcentrifuge tubes using separate pellet pestles. Grind for approximately 1 minute. The plant tissue sample should become green when it is fully ground.
4. Add 900 mL of Edward's buffer to each tube containing the ground sample. Grind briefly to remove tissue from the pellet pestle and to liquify any remaining pieces of sample.
5. Vortex the tubes for 5 seconds, by hand or machine. Boil the samples for 5 minutes in a water bath or heating block. Note: Be sure to monitor the samples as the eppendorf tube lids may open as the tubes heat.
6. Microcentrifuge the tubes containing the ground plant tissue or food sample for 2 minutes. After 2 minutes any insoluble material should form a tight pellet at the bottom of the tubes.
7. Transfer 350 mL of each supernatant to a fresh tube. The supernatant contains the desired DNA; make sure not to disturb the pelleted material when transferring the supernatant. (This is best accomplished by pipetting several times using a medium micropipet set at 100 mL.)
8. Add 400 mL of isopropanol to each of the DNA-containing supernatants. Mix, and leave at room temperature for 3 minutes. This step precipitates the DNA.
9. Microcentrifuge the tubes with the isopropanol and supernatant for 5 minutes with the hinge of tubes pointing outward.
10. After centrifugation, the pellet should be located at the bottom-side of the tubes underneath the hinge. It may be teardrop shaped or appear as small, scattered granules. Don’t be concerned if you can’t see a pellet. Carefully pour off the supernatant, then completely remove the remaining liquid with a medium pipet set at 100 mL.
11. Air dry the pellets for 10 minutes to remove any remaining isopropanol.
12. After drying, resuspend each DNA pellet in 100 mL of TE buffer. Pipet in and out, taking care to wash down the side of the tube underneath the hinge, where the DNA has accumulated during centrifugation.