2. Abstract
Even with GMOs being one of the most talked about and controversial topics in the food and
agricultural world today, it is impossible for the average consumer to see whether or not their
food contains GMOs or not. Even in laboratories discovering such information used to be a long
and extensive process. But with the help of some modern laboratory technology like gel
electrophoresis and thermocyclers, the ability to test a food for GMOs has become possible for
common laboratories to test. Our class decided to take advantage of such technology and used it
along with restriction enzymes to determine whether or not four common snack foods contained
plant DNA and/or GMO DNA. It was found that all four foods tested contained plant DNA.
While only two of the four foods contained GMO DNA.
Introduction
Arguably, one of the most controversial topics in modern agriculture is the use of genetic
engineering. Yet while genetically modified (GM) foods are one of the hottest topics today, it is
also one of the unknown factors in many foods. It is estimated that 60-70 percent of every food
at the grocery store contains at least one GM ingredient (Bryne, 2010). None of these foods,
though, are labeled for GM foods by their manufacturers due to the FDA not mandating it. But
what exactly are GM foods, why are they made, how are they made, and how they can be
detected?
In short, GM foods are any foods which have had foreign genes inserted into them. These foreign
genes, which come from other plants or animals, give the organisms an enhanced genetic code
which modifies the proteins made by the plant. Some foods, for example have foreign genes
which allow them to grow in colder temperatures where before they were strictly warm weather
plants (Center for Food Safety, 2013). This is just one of the many reasons GM is used with
foods today. Historically, the first genetically modified food (or GM food) marketed in the US
was the Flavr Savr tomato in 1994- which allowed the tomato to delay ripening after picking
(James & Krattinger, 1996). Since then, the industry for GM foods has exploded with the
company Monsanto leading the industry. As of 2013, roughly 85% of corn, 91% of soybeans,
and 88% of cotton produced in the United States are genetically modified (Center for Food
Safety, 2013). According to the FDA and other government organizations which approve these
foods, however, there is a reason behind such high percentages. GM foods are said to give a
multitude of benefits to the general public and the environment. Some of these benefits include
more nutritious food, higher crop yield, a decreased use of pesticides, and disease resistant plants
(National Institutes of Health, 2012).
But there is another side to the GM food controversy. Aside from potential benefits of GM foods,
there are also potential risks to consider. Such risks could include GM foods having potential
genetic changes making them unsafe for human consumption and the cause of so called super
weeds which have become resistant to many pesticides (National Institutes of Health, 2012).
3. Without the labeling, it is extremely difficult to know whether or not a product contains GMOs.
While logical guesses are possible by comparing the main ingredients of a food with common
GMOs crops such as corn and soybeans, they are only guesses nonetheless. However, there is
actually a way to detect for the presence of GMOs using modern laboratory techniques.
PCR is a molecular technique used to amplify a specific sequence of DNA defined by a set of
primers producing millions of copies of that sequence or gene. It does this by rapidly increasing
the temperature denaturing the DNA, then lowering it again to allow the primers to anneal to the
denatured DNA. The PCR machine then raises the temperature one last time to the optimum
temperature for replication forcing the DNA to replicate. Usually, there is around 35-40 cycles
per experiment. Gel electrophoresis usually follows PCR amplifications. It is used to separate the
amplification product in each sample based on size and charge revealing bands of DNA and their
molecular weight. Using these techniques, we conducted an experiment to discover whether or
not four common foods contained either GMO DNA or came from a GM plant. Due to the four
foods tested--Cheddar Sun Chips, Kettle Corn Chips, Nilla Wafers, and Frito Corn Chips—I
expect all four to contain both GMOs.
Materials and Methods
Two small electronic scales were taken from the back room along with eight weighing trays. The
scales were then balanced to ensure the most accurate results and a weighing tray was set on the
scale and zeroed out to exclude the weight of the tray from the sample. Our negative control
(Bio-Rad Oatmeal) was then weighed out for each of the four groups at the following masses:
1.65 g, 1.5 g, 1.56 g, and 1.52 g. Each group then placed their negative control in a pestle and
mortar (1 for each group). The negative controls were done first in order to prevent cross
contamination of possible GMO DNA in our test samples. The mass of the negative control was
then multiplied by five to determine the amount of DNase water to add. Using this equation
groups 1-4, respectfully, added, 8.25 ml, 7.5 ml, 6.45 ml, and 7.6 ml of DNase water to pestle
and mortar. The mixture was then ground up for three minutes or until the oatmeal was broken
apart. Each group then added the same amount of DNase water as they did the first time once
again. The mixture was subsequently ground up for another 3-5 minutes, or until the oatmeal was
completely mixed with the DNase water. Following the negative control being mixed in, 50ul of
the mixture was pipetted to a microfuge tube filled with 500ul of Instagene. Each group
performed this step but with their own sample.
The pestle and mortars were then washed out with water and a detergent to attempt to get rid of
any trace of the negative control. The scales were zeroed out with a weighing dish again. In
chronological order, each group then weighed out their sample to be, 1.5 g, 1.5 g, 1.56 g, and
1.84 grams. Using the same equation mentioned above each group then added, 7.5 ml, 7.5 ml,
7.8 ml, and 9.2ml of DNase water to the pestle and mortar along with their sample food. The
order of the previous amounts, from left to right, goes group 1 to group 4. The mixtures were
then ground up for a few minutes. The four groups then added the same amount of DNase water
4. to the mixture ground it up for a couple more minutes until their sample was mixed with the
DNase water. 50ul of the mixture was then pipetted into a microfuge tube with 500ul of
Instagene. All eight tubes (two from each group) were then placed on a 95 degree Celsius water
bath for five minutes before placed in a centrifuge for five minutes at max speed.
After making the samples, two PCR master mixes were made using the following ingredients:
Mg2+
, dNTPs, PCR water, primers, buffer, and a taq enzyme. The difference between the two
types is that the primers for the plant master mix are made to target specific plant DNA; while
the GMO master mix primers are made to target GMO genes. Six PCR tubes were then gathered
and numbered 1-6. They were then filled according to table 1. The samples were then amplified
using polymerase chain reaction inside a thermo cycler set to the following settings:
94o
C for 30 seconds
59o
C for 30 seconds
72o
C for 1 minute
Repeat 40 times
Following the samples being run through PCR, they were removed from the machine and set into
tube holders. Two, 3% agarose gels were then made with 13 cells each. To make each gel, we
combined 1.5 grams of gel agarose power with 50ml of 1x TAE buffer in a 200ml beaker. The
1x TAE buffer was made by adding 20ml of 50x TAE buffer with 980ml of dH2O. The mixtures
were then stirred until the agarose was completely dissolved and the mixtures were both cloudy
and opaque. With the microwave at low power, the two beakers were placed in it and then
cooked for 2 minutes. When the gels were removed the mixture had turned translucent. At this
stage, ethidium bromide was added to each gel. The gels were then placed back in the microwave
for 1 minute. Following the second microwave cycle, the gels were poured into two casting trays.
The casting trays themselves had to be taped due to the sides being open which would allow the
agarose to pour out the sides. The two gels were then placed in the fume hood and a 13 cell comb
was placed into each one. The gels were then given time to polymerize in the fume hood.
Following the polymerization, two groups were assigned to each gel. The tape was then taken off
the casting trays and the gels were placed in the gel electrophoresis machine. 1x TAE buffer was
then added to each machine until the buffer level was higher than the cells. Before the samples
were loaded, however, 10ul of Orange G loading dye was added to each of the combined 24
samples. Each of the 13 cells in each gel was then filled 10ul of sample using a micropipette. For
both gels, lanes 1-6 and 8-13 contained the samples in numerical order. Lane 7 contained the
PCR molecular weight ruler. Following loading, each gel was then analyzed through gel
electrophoresis for 30 minutes at 100 volts. The gels were then removed at the end of the 30
minutes and examined under a UV light.
5. Results
In figure one, as seen below, two separate gels were made each containing two groups each. For
Preston and Alica’s group (lanes 1-6) their test sample, Frito Corn Chips, tested positive for both
plant and GMO DNA. Lanes 8-13 contained Megan and Echoe’s groups’ experiment. Their test
food was Nilla Wafers and it tested negative for GMO DNA but positive for plant DNA. In the
second gel from lanes 14-19 Casy and Derek’s food sample, Kettle Corn Chips, it too was
negative for GMO DNA but positive for plant DNA. Tarence and I’s sample, Cheddar Sun
Chips, was analyzed in lanes 21-26 then. The Sun Chips were positive for both GMO DNA and
plant DNA.
For each sample which tested positive for GMOs as well as the positive control, the bands which
appeared were 200 base pairs in length. If a sample was positive for plant DNA or was the non-
GMO negative control, bands appeared at 455 base pairs. Primer dimers also occurred in lanes 4,
6, 11, 13, 17, 19, and 26.
Discussion
Out of the four foods tested, only two of them contained GMO DNA, Sun Chips and Frito Corn
Chips. As stated in my hypothesis I expected the Sun Chips and Frito Corn chips to contain
GMO DNA, which they did. However, the other two samples, Nilla Wafers and Kettle Corn
Chips, were negative for GMO DNA. To me, this came back as surprising due to those two items
being derived from big time companies which I assumed would implant GMOs in their products.
Due to this I hypothesized that all four food samples would contain GMO DNA. All four
samples did contain plant DNA, though. This means that the plant(s) the food item(s) came from
were made using genetic engineering and therefore were GM crops.
In order to verify the data found for each sample was accurate, we used the DNA ladder found in
lanes 7 and 14 along with the positive control(s). For both groups one and four where the food
samples tested were positive for GMOs, the band found in those lanes (4&24) aligns with both
the GMO positive control (GMO master mix) and the DNA molecular weight ruler at 200 base
pairs. The same pattern occurs for the plant DNA which tested positive for all four groups (lanes
3, 10, 16, and 23). Each of them aligns with the GMO positive control (plant master mix) and the
molecular weight ruler at 455 base pairs.
As stated in the results, primer dimers were seen in lanes 4, 6, 11, 13, 17, 19, and 26. These faint
bands, known as primer dimers, occur when two primers anneal with each other during PCR and
then are elongated by the taq enzyme. When separated by gel electrophoresis, these two primers
are pulled by the positive electrode beyond the DNA ladder and appear as a very faint band.
One of the few problems we encountered with this experiment was the non-GMO negative
control band not showing up in lanes 1 and 14. Due to it being a non-GMO negative control
mixed with the plant master mix, the primers in the master mix were targeting plant DNA. So the
6. lane is supposed to be negative for GMO DNA which would be found at 200 base pairs, but
positive for plant DNA due to it being the target of the primers. However, the plant DNA failed
to show up in either of the two gels meaning experimental error must have happened. There are
multiple possible answers to this is, one of them being the negative control oatmeal was no
ground up enough. Not grinding the oatmeal up enough would not release the DNA. Therefore
there was little to no DNA pipetted into the Instagene tubes. There could have also been too
much of the sample pipetted into the Instagene tubes (supposed to be 50 ul). Having too much
sample would require the Instagene to theoretically ‘work harder’ which would cause the
DNases to not be destroyed in the water bath. Failure to destroy the DNases will result in our
samples’ DNA being destroyed. Leaving nothing for PCR to amplify and gel electrophoresis to
separate.
7. 1 20ul Non-GMO food control 20ul plant master mix
2 20 ul Non-GMO food control 20ul GMO master mix
3 20ul test food 20ul plant master mix
4 20ul test food 20ul GMO master mix
5 20ul GMO positive control 20ul plant master mix
6 20ul GMO positive control 20ul GMO master mix
Table 1. The amount of liquid, in microliters, which went into each of
the six PCR tubes.
Figure 1. PCR amplification of test samples for the presence of plant- or GMO-DNA sequences. Negative control
(lanes 1&2, 8&9, 14&15, 21&22), a DNA ladder (lanes 7&20), and positive control (5&6, 12&13, 18&19, 25&26)
samples were processed and amplified in tandem with each test sample. The DNA ladder was composed of five
bands measuring 100, 200, 500, 700, and 1000 base pairs in length. The food products tested in the experiment
included Frito Corn Chips (lanes 3&4, group 1), Nilla Wafers (lanes 10&11, group 2), Kettle Corn Chips by
PopCorners (lanes 16&17, group 3), and Cheddar SunChips (23&24, group 4). For each pair of samples (e.g., 1&2,
8&9 etc.) the first lane was amplified using plant DNA-specific primers while the second lane was amplified using
GMO DNA-specific primers.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
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