Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Genetic modification of foods has been a controversial issue due to debated concerns over health and environmental safety. This experiment demonstrates technical understanding of how food DNA is genetically identified, allowing for educated decision making about the safety and potential dangers of using genetically modified organisms (GMOs) in food supplies.
Polymerase Chain Reaction (PCR) is used to amplify food DNA to test for the presence of genetically modified DNA in food products. Presence of specific DNA bands is detected by using gel electrophoresis to pull extracted food DNA through a 3% agarose gel, a concentration dense enough to separate the bands of DNA containing the genetically modified DNA. Several controls are used in the electrophoresis procedure to ensure DNA is successfully extracted from test foods (plant primer), and to provide known examples of both genetically modified DNA (purchased genetically modified DNA) and non-genetically modified DNA (purchased certified non-GMO food control).
Polymerase Chain Reaction (PCR) identifies sequences of DNA that have been inserted into the GM plant. In contrast to proteins, DNA is a relatively stable molecule, thus DNA fragments can be isolated from highly processed goods and are sufficiently intact to be amplified by PCR. Genetic engineers use only a small number of regulatory sequences (promoter and terminator sequences) to control the expression of the inserted genes, and so these sequences are common to the majority of GM crops. The two sequences identified in this procedure are two of the most common regulatory sequences, 35S promoter gene from cauliflower mosaic virus (CaMV) and the nopaline synthase (NOS) terminator gene from Agrobacterium tumefaciens.
PCR involves repetitive cycles, each consisting of template denaturation, primer annealing, and extension of the annealed primer by Taq DNA polymerase. Once DNA is extracted from food, a thermal cycler is used to rapidly manipulate temperature, causing the stages of the PCR cycles.
The denaturing stage occurs when samples are rapidly heated to 94 °C, causing the DNA strands to separate. Rapid cooling to 59 °C allows primers to anneal to the separated DNA strands, then reheat to 72 °C for Taq DNA polymerase to extend the primers, making complete copies of each DNA strand and completing one thermal cycle.
The amplified DNA can then be run through an agarose gel with electrophoresis to separate the DNA into visible bands for identification of the 35S promoter gene and the NOS terminator. Amplified DNA is loaded into wells at one end of the gel, using a purchased loading dye that helps weigh down the sample to prevent dissolution into the surrounding buffer. The loading dye also provides a visual, so the movement of DNA can be seen during electrophoresis in the dye “front.” The electrophoresis process works by using an electric current separated into cathode and anode ends. DNA is loaded into the gel on the end closest to the cathode side of the chamber, and the negative charge of DNA is attracted to the anode end of the chamber and is pulled through the agarose. Larger sequences of DNA (increased number of base pairs) cannot move as easily through agarose and will separate out early, while the smaller sequences are able to travel farther down the gel toward the anode end.
A staining process helps by binding to the DNA in order to add contrast between the background gel and the banks of DNA for better visualization of results. Using provided known locations for the different sizes of DNA sequences each test gel can be analyzed for the presence or absence of the 35S promoter and NOS terminator genes.
Controls are used to ensure DNA is extracted properly and to provide a comparison to the test food samples. Purchased plant primer is added to each sample to provide nucleic acids common to all plants. This allows for a quality control check on the DNA extraction process, because any plant DNA extracted should be extended with this primer during PCR and should also be seen on the gel after electrophoresis is complete. Purchased primer for the 35S and NOS genes are used as a positive control to provide DNA bands on the gel for genetic modification. If the GMO-positive template control does not amplify, there is a problem with the PCR reaction and a GMO-negative result from the test food cannot be trusted. A certified non-GMO food product is also purchased and used as a negative control to show what DNA separation looks like when no genetically modified material is present.
1. Extraction of DNA From Food Samples
- Add 500 µL of purchased PCR mix matrix to each of the 2 screw-cap tubes using a transfer pipet or 200-1,000 µL adjustable-volume micropipette. Pipette up and down with the pipet between each aliquot to evenly mix the PCR matrix.
- Label one screw-cap tube “non-GMO” and the other “test”.
- Weigh out 0.5 g of certified non-GMO food and put it into the mortar.
- Add 2.5 mL of distilled water and grind with pestle for 2 min to form a slurry.
- Add another 2.5 mL of distilled water and continue grinding with pestle until slurry becomes smooth enough to pipet.
- Pipet 50 µL of ground slurry to the screw-cap tube containing 500 µL of PCR premix labeled “non-GMO” using the 50-µL mark on a graduated pipet. Recap tube.
- Repeat steps 1.3–1.6 to prepare the test food sample.
- Pipet 50 µL of ground test food slurry to the screw-cap tube labeled “test”. Recap tube.
- Vortex the non-GMO food and test food PCR tubes for 1 min and place tubes in 95 °C water bath for 5 min. If no vortex is available, flick the tube several times to mix before placing in water bath.
- Place tubes in a centrifuge for 5 min. A solid pellet should form at the bottom of the tube. If pellet is not formed after 5 min, centrifuge again for 2 min intervals until pellet forms.
- Tubes can be used immediately for PCR or stored in a refrigerator for up to 1 week.
2. Setting Up PCR Reactions
- Number PCR tubes 1–6 and initial them. The numbers should correspond to the following tube contents listed in Table 1.
- Place each labeled PCR tube in a microtube holder with caps open.
- Using a fresh tip for each addition, add 20 µL of the primer indicated on Table 1 to each PCR tube. Cap tubes.
- Using a fresh tip for each tube, add 20 µL of the DNA sample indicated on Table 1 to each PCR tube, being sure to pipette only from the supernatant and avoiding the solid pellet at the bottom of the tubes.
- After each DNA sample is pipetted into the corresponding PCR tube, use pipette to mix DNA and primer by pipetting gently up and down; recap tubes.
- Place PCR tubes in thermal cycler and program the cycler for:
Initial Denature: 1 cycle at 94 °C for 2 min.
Amplification: 40 cycles at 94 °C for 1 min (denature), 59 °C for 1 min (annealing), and 72 °C for 2 min (extension).
Final Extension: 1 cycle at 72 °C for 10 min.
Hold: 4 °C indefinitely.
3. 3% Agarose Gel Preparation
- Using lab or masking tape, securely tape off the open ends of the gel tray. Ensure tape is sealed to the edges of the tray to prevent molten agarose from leaking out.
- Weigh out 3 g of agarose into a 250-mL or larger Erlenmeyer flask.
- Add 100 mL of 1x TAE buffer (purchased or prepared from concentrate).
- Using a magnetic hot plate with a stir bar, heat the beaker until agarose is completely dissolved in the buffer and the solution is boiling and turns clear. Alternatively, a microwave oven can be used by placing beaker in oven and heating for 30 s intervals, using a stir rod to mix every 10 s, repeating until mixture is boiling and turns clear.
- Invert a 50-mL Erlenmeyer flask into the opening of the 250-mL flask to act as a reflux, preventing agarose solution from evaporating.
- Allow agarose solution to cool to 60 °C, and pour 30-50 mL into each taped gel tray.
- Place a gel comb into the first pair of notches to create gel wells.
- Allow gel to cool completely before removing tape and comb. Gel will solidify and turn from clear to cloudy when ready, approximately 10-20 min.
4. Electrophoresis of PCR Products
- Place a pre-made 3% agarose gel onto a gel tray or use a gel tray that has been used to cast a poured 3% agarose gel.
- Slide gel tray into the electrophoresis chamber with the wells closest to the cathode (black) end.
- Pour 1x TAE buffer into the chamber, enough to have 2 mm of buffer above the top of the gel tray.
- Obtain PCR tubes from the thermal cycler and place in microtube holder.
- Using a fresh pipette tip each time, add 10 µL of purchased Orange G loading dye (LD) to each sample and mix well.
- Load 20 µl of the molecular weight ruler and 20 µL of each sample into the gel in the order indicated (Table 2).
- Run gel electrophoresis for 30 min at 100 V.
- Remove gel tray from chamber and slide gel to remove from tray. Place gel in a staining tray.
- Immerse gel in purchased DNA gel stain for 5 min, carefully shaking tray to help distribute the stain throughout the gel.
- Transfer the gel into a washing container and rinse with tap water (40-55 °C) for approximately 10 s.
- Destain by washing 3x in warm tap water for 6 min each with gentle shaking for best results. If necessary, continue destaining in warm water until the desired contrast is reached.
|Tube number||Primer||Dna Sample|
|1||20 µL Plant primer (green)||20 µL Non-GMO food control DNA|
|2||20 µL GMO primer (red)||20 µL Non-GMO food control DNA|
|3||20 µL Plant primer (green)||20 µL Test food DNA|
|4||20 µL GMO primer (red)||20 µL Test food DNA|
|5||20 µL Plant primer (green)||20 µL GMO positive control DNA|
|6||20 µL GMO primer (red)||20 µL GMO positive control DNA|
Table 1. List of the appropriate tube numbers, primers, and DNA samples.
|Well 1||Sample 1 Non-GMO food control with plant primers 20 µL.|
|Well 2||Sample 2 Non-GMO food control with GMO primers 20 µL.|
|Well 3||Sample 3 Test food with plant primers 20 µL.|
|Well 4||Sample 4 Test food with GMO primers 20 µL.|
|Well 5||Sample 5 GMO positive DNA with plant primers 20 µL.|
|Well 6||Sample 6 GMO positive DNA with GMO primers 20 µL.|
|Well 7||PCR molecular weight ruler 20 µL.|
|Well 8||Leave empty.|
Table 2. The appropriate order to load 20 µL of the molecular weight ruler and 20 µL of each sample into the gel.
Genetically modified foods are products which contain an ingredient or ingredients whose DNA has been specifically altered. Presence of these modifications can be detected using a technique called the polymerase chain reaction.
Genetic modification of foods has been a controversial issue due to debated concerns over health and environmental safety. The ability to detect genetically altered DNA in food samples of interest allows for educated decision-making about the safety and potential dangers of using genetically modified organisms, or GMO's, in food supplies.
The polymerase chain reaction, or PCR, is used to amplify food DNA to determine the presence or absence of genetically modified sequences. Gel electrophoresis then pulls the amplified DNA through an agarose gel matrix and separates DNA bands of different sizes that correspond to modified or non-modified markers. These are then compared to control bands from foods known to contain GMO's, or known to be modification free.
This video will illustrate the principles behind detecting genetically modified DNA from food samples, how to extract DNA and amplify markers used in the genetic modification process, and how the presence or absence of GMO's in food samples can be established.
Polymerase chain reaction identifies sequences of DNA that have been inserted into the genetically modified food. DNA is a relatively stable molecule, so viable DNA fragments suitable for amplification can be isolated even from highly processed goods, like corn chips or vegetable burgers.
A small number of regulatory DNA sequences are used to control the expression of inserted genes, so these sequences are present in the majority of GM crops. This procedure identifies two of the most commonly used, the 35S promoter gene and the nopaline synthase terminator gene. The 35S sequence is a strong promoter, and when attached to an inserted gene will drive constant, high levels of expression. The nopaline synthase terminator is included to stop transcription of the inserted gene at the desired endpoint.
PCR involves repetitive heating and cooling cycles in a thermal cycler, a machine that tightly controls the temperature of PCR reaction tubes. Heating the sample to 94 °C causes DNA strands to denature and separate. Rapid cooling to between 45 and 65 °C allows primers to anneal to the separated DNA strands. Finally, reheating to 72 °C allows the Taq polymerase enzyme to extend the primers and complete duplication of the target region.
Purchased plant primer is added to each sample, and will amplify in samples containing plant DNA. GMO positive templates for 35S and NOS are used to provide a positive control for GMOs. A certified non-GMO is used as a negative control. If any of these control reactions show unexpected bands, the results of test samples cannot be trusted.
Amplified DNA is run through an agarose gel by electrophoresis. PCR products are mixed with a dye and loaded into wells. DNA is negatively charged, and when loaded into the gel at the cathode end of the chamber will move toward the anode end when current is applied. Larger DNA fragments cannot move as easily through the gel matrix so will stay closer to the cathode, while smaller sequences move toward the anode end, resulting in separation of differently sized DNA into distinct bands.
After electrophoresis, gel staining helps visualize separated DNA bands.
Now that we are familiar with the principles behind GMO detection and the use of PCR and electrophoresis for GMO identification, let's take a look at how this can be carried out in the laboratory.
Once the food products of interest have been identified, analysis can begin. To extract the DNA, take two clean screwcap tubes, and into each of these transfer 500 μL of purchased DNA isolation reagent, making sure to pipette the mix up and down between aliquots to evenly mix the reagent. Label one of the tubes "non-GMO", and the other "Test".
Next, weigh out 0.5 g of certified non-GMO food, and place into a clean mortar. Add 2.5 mL of distilled water, and grind with the pestle for 2 min to form a slurry. Add another 2.5 mL distilled water and continue grinding the slurry until it becomes smooth enough to pipette.
Pipette 50 μL of the known non-GMO slurry to the "non-GMO" labeled screwcap tube containing the DNA isolation reagent. Now add 50 μL of the test food sample slurry to the screwcap tube marked "Test". Vortex the two tubes containing the food samples and DNA reagent for 1 min. Next, place the tubes in a water bath at 95 °C for 5 min. Finally, place the tubes in a centrifuge for 5 min. Upon examination, a solid pellet should be formed at the bottom of the tube. If a pellet is not formed after 5 min, centrifuge again for 2-min intervals until a pellet forms. Samples can now be used immediately for PCR, or stored in a refrigerator for up to 1 week.
First, number six PCR tubes. These numbers will correspond to the tube contents listed in the Table. Place each of the labeled PCR tubes in a microtube holder with caps open.
Using a fresh tip for each addition, add 20 μL primer indicated in the Table to each PCR tube. Next, add 20 μL of the DNA samples indicated in the Table to each PCR tube. Pipette up and down to mix. For pelleted samples, be sure to pipette only from the supernatant, and avoid the solid pellet at the bottom of the tubes. Finally, place the PCR tubes into a thermal cycler and program it to cycle through the heating and cooling steps indicated in the table.
Place a 3% agarose gel into the electrophoresis chamber with the wells closest to the cathode end. Add TAE buffer into the chamber to cover 2 mm above the gel tray. Collect the PCR tubes from the thermocycler and place them in a microtube holder. Using a fresh pipette tip each time, add 10 μL of purchased DNA loading dye to each sample and mix well.
Using a fresh tip each time, load the wells of the agarose gel with 20 μL of molecular weight ruler into one well, and 20 μL of each sample into subsequent wells, as indicated in this table. Set the electrophoresis chamber to run at 100 V for 30 min.
Once the gel has finished running, carefully unplug the gel chamber, remove the tray, and slide the gel into a staining tray. Immerse the gel in purchased 100x gel stain for 5 min shaking the tray gently to distribute the stain. Once stained, transfer the gel into a washing container and rinse with tap water for approximately 10 s. Destain by washing three times in warm tap water for 3 min each, each with gentle shaking for best results. If necessary, continue destaining in warm water until the desired contrast is reached.
The presence or absence of a 200 bp band in lane 4 indicates whether the test food contains GMOs. The plant primers determine whether plant DNA was successfully extracted from the sample. The non-GMO food control is an indicator of false positive results, should they occur. If the non-GMO food control comes out as positive, it means the PCR was contaminated at some point. The GMO-positive template control is an indicator of false negatives. If the GMO-positive template control does not amplify, there is a problem with the PCR reaction and a GMO-negative result from the test food can’t be trusted.
Gels can be placed on a white or yellow paper to provide contrasting background to highlight DNA bands, or increased contrast can be achieved using a UV light box.
The ability to test foodstuffs or products for genetically modified ingredients is pertinent to a number of scientific or regulatory applications.
There is some evidence that transgenic DNA from genetically modified crops can become introgressed into the genomes of wild crop populations. For example, pollinators may facilitate this transfer by fertilizing wild-type plants with pollen from a genetically modified source. This is a concern, as the impacts of the spread of these inserted genes on ecosystems as a whole are not well understood. PCR testing of wild populations can provide data on the potential spread, or successful containment, of genetic inserts.
Lack of labeling, or incorrect labeling of genetically modified ingredients can be a consumer concern. This may be due to the consumer's preference of consumption, or potential introduction of allergens during the GM process, though the latter is controversial. In some countries, genetically modified ingredients are required to be listed on food packaging. Regulatory boards in such countries may use PCR testing to check the accuracy of food labeling.
Where the genetic modification introduced to a crop confers pesticide resistance, some studies have raised concerns about the production of "super-weeds". Essentially, these can be any plant not initially targeted for modification that obtains the resistance to pesticide through mechanisms such as introgression or hybridization. These plants may then be able to spread more successfully, and be harder to control than those without the insert. PCR testing for genetic modification can help to highlight and track such potential populations to determine if this spread could prove problematic.
You've just watched JoVE's introduction to Testing for GMO Foods. You should now understand the principles behind identifying genetically modified foods using PCR, how to extract and amplify DNA from food, and how to determine if your food sample has been genetically modified. Thanks for watching!
After destaining, gels can be analyzed by looking at test food lanes (Table 3) to determine if the DNA bands for the 35S promoter and NOS terminator genes are present in the known locations on the gel. Placing the gel on a UV light box can help provide increased contrast (Figure 1). Alternatively, gels can be placed on white or yellow paper to provide a contrasting background to highlight DNA bands (Figure 2).
Figure 1. A destained gel showing separated bands of DNA. Agarose gel following agarose gel electrophoresis on UV light box.
Figure 2. Diagram of known locations for the 35S promoter and NOS terminator DNA. The presence or absence of a 200 bp band in lane 5 indicates whether or not the test food contains GMOs.
|Lane 1: Non-GMO food with plant primers||455 bp|
|Lane 2: Non-GMO food with GMO primers||No band|
|Lane 3: Test food with plant primers||455 bp|
|Lane 4: Test food with GMO primers||200 bp or no band|
|Lane 5: GMO-positive template with plant primers||455 bp|
|Lane 6: GMO-positive template with GMO primers||200 bp|
|Lane 7: PCR molecular weight ruler||1,000, 700, 500, 200, 100 bp|
Table 3. PCR Sample Band Sizes (base pairs (bp)).
Applications and Summary
Polymerase Chain Reaction (PCR) is used to amplify DNA, allowing for a wide range of DNA lab testing. One area of testing now possible with PCR is to identify GMOs by testing for presence or absence of the DNA sequences used in the genetic modification of food crops. Typically, a crop is genetically modified to confer an advantage against natural deterrents to ideal yields, e.g. pests (Figure 3), diseases, drought conditions (Figure 4), etc. Because the advantage is gained by inserting genetic material from a different species into the crop plant’s own DNA, potential human health and environmental risks have been identified with the use of GMOs. One environmental concern is the ability of the genetically modified DNA to be exchanged unintentionally through pollination processes, which could lead to genetically modified DNA entering the genomes of crops intended to be sold as non-GMOs.
Figure 3. Larvae of Colorado beetle, devouring leaves of a potato.
Figure 4. Corn destroyed by drought.