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Isabella Roig
BIO-300-001
16 October 2013
Erica Brown
Bella Muller, Maia Plesent, Mariam El-Magbri
Detecting Genetically Modified Foods by PCR
Abstract
In this experiment, the PCR technique was observed and used to identify whether DNA
of various food and plant products has been modified. Positive and Negative Controls, Roundup
Ready/Knockout soybean and Wildtype soybean were used as to demonstrate what genetically
modified DNA looks like and what is not look like in terms of particular sequences. After
viewing the H7 gel, it was concluded that Food Product #2 and Wildtype soybean were not
genetically modified. It was also concluded that Food Product #1 and Roundup Ready/ Knockout
were genetically modified.
Introduction
Genetically modified organisms are very common nowadays in the United States. Over
60% of food is genetically modified. To genetically modify, is defines as, to alter the DNA in an
unnatural way. One of the most common genetic alterations is to add the glyphosate-resistance
gene. This gene protects the food from Roundup, a common herbicide. Genetically modified
foods have sequences added to their DNA strands that code for resistance against pesticides or
code specific characteristics. (Huang, et al. 2013).
The purpose of this experiment was to learn PCR technique as a way of identifying
whether DNA from various food and plant sources is modified. PCR is used to amplify a specific
section of DNA. The 35S promoter drives the expression of the glyphosate-resistance gene. To
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look for this, 35S primer was added to all the samples. If the 35s promoter appears in gel by
electrophoresis, then it can be concluded that product is genetically modified. PCR yields the
DNA fragment at 162 base pairs, which is where the 35S promoter amplifies on the DNA strand.
To ensure that the PCR process is successful for each sample, tubulin was used as a positive
control because it is a protein found in all food and soy products.
In this lab, Roundup Ready soybean, Wildtype soybean, Chex cereal, and rice cake were
observed to identify were products had been genetically modified. Roundup Ready soybean was
used as a positive control because it is known that that soybean was genetically modified to be
“ready” to combat the pesticide Roundup. Wildtype was used as a negative control because it is
known that that soybean was organic and not genetically modified. Using these two controls and
tubulin, this experiment had parameters to measure the success or failure and accuracy of the
PCR technique. It is hypothesized that only products that were genetically modified will have a
band located at 162bp in the gel. It is hypothesized that Chex cereal and rice cake are genetically
modified and will have a band located at 162bp. It was also hypothesized that all samples with
the tubulin primer will have a band located at 187bp.
Materials and Methods
In a 1.5mL tube, place a few pieces of Wildtype soybeans that have been cut to
approximately ½ inch in diameter. In another tube, the same was done with Roundup Ready
soybeans. The tubes were labeled with each soybean type and the group number. Two types of
dry food product were used in this experiment. The first food product was crushed into a powder.
It was added to a 1.5mL tube to the 0.1mL marking. The same was done for the second food
product. These tubes were labeled with the group number and either FP1 or FP2. 100L of
Edward’s buffer was added to the four tubes. A clean plastic pestle was used to grind up the food
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or plant product with the buffer for one minute. 900L of Edward’s buffer was added to each
tube. The tubes were placed by hand on the vortex for five seconds. The samples were boiled for
five minutes in a heating block. After placing the tubes in a balanced configuration in a
microcentrifuge, they were spun for two minutes. 350L of the supernatant from each tube was
transferred into a separate fresh 1.5mL tube. The new tubes were again labeled with group name
and the plant product or food product. 400 L of isopropanol was added to each tube of
supernatant. The tubes were inverted several times to mix the contents. The tubes were incubated
at room temperature for three minutes. The tubes were placed in a balanced configuration with
the cap hinges pointing outward in a microcentrifuge and spun for five minutes. The supernatant
from each tube was poured out and discarded. The remaining liquid was completely extracted
using a medium pipet set at 100L. The air pellets were left to dry with the tube caps open for
ten minutes. The isopropanol evaporated. 100L of TE/RNase A buffer was added to each tube.
The nucleic acid pellet was dissolved by pipetting in and out. The TE/RNase A solution was
incubated for five minutes. The tubes were placed in the microcentrifuge to pellet any material
that did not go into solution.
After obtaining four PCR tubes with Ready-To-Go PCR Beads, they were labeled “35S
FP1”, “35S FP2”, “35S RR” (for Ready Round-Up soybean), and “35S WT” (for Wildtype
soybean). Using a micropipette, 22.5L of the 35S primer/loading dye mix was added to the four
tubes. With a fresh tip, 2.5L of Food Product #1 DNA was added to the tube labeled “35S
FP1”. The same was done for the second food produce, Roundup Ready soybean, and Wildtype
soybean in their respective tubes.
Four more PCR tubes with Ready-To-Go PCR Beads were obtained. They were labeled
“T FP1”, “T FP2”, “T RR” (for Ready Round-Up soybean), and “T WT” (for Wildtype
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soybean). Using a micropipette, 22.5L of the tubulin primer/loading dye mix was added to the
four tubes. With a fresh tip, 2.5L of Food Product #1 DNA was added to the tube labeled “T
FP1”. The same was done for the second food produce, Roundup Ready soybean, and Wildtype
soybean in their respective tubes. The samples were stored on ice until the entire lab was ready to
being thermal cycling.
The TA followed the following thermal cycle: Denaturing- 30 seconds at 94°C,
Annealing- 30 seconds at 60°C, Extending- 30 seconds at 72°C. After the thermal cycle was
completed, the samples were stored at -20°C until the following week.
A gel holder was snugly placed into the preparation rack with a comb near one end of the
gel space. 1.0g of agarose was placed in a flask with 50ml of 1x TBE buffer and swirled to mix.
The flask was heated in the microwave for 1-2 minutes and then left to cool on the bench.
Ethidium bromide was added to the flask. Then, the contents of the flask were poured in the gel
rack. The gel cooled and solidified for twenty minutes on the bench. The comb was then
removed. The solidified gel was place in the electrophoresis chamber after being removed from
the preparation rack. TBE buffer was added until the top of the gel was just barely covered by
liquid. The 20L of each sample were then loaded into separate wells of the gel. The cover was
placed on the apparatus so that the loaded wells were closest to the black electrode. All group
members had checked twice that the apparatus was plugged into the power supply and set up
correctly, red to red and black to black, before turning on the power supply at 120V. After the
dye in the gel had migrated at least halfway down the gel, the power supply was turned off, the
apparatus was disconnected, and the lid was removed. The gel was placed on a piece of
aluminum foil can set on the UV light to be viewed.
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Results
Figure 2 shows the results of this particular experiment. No bands were visible in any of
the wells under the UV light, except for the marker. These results could not be analyzed, so
results from a similar experiment were used for analysis. The picture in Figure 1 was not
produced by this particular experiment, but it shows a similar result to would have been seen by
this particular experiment. Because this picture is not from this experiment, another genetically
modified soybean, Knockout, had been used rather than Ready Roundup.
Well 3, Wildtype soybean 35S, being the negative control, did not have a band at 162bp,
indicating at it was not genetically modified. Knockout, the positive control, had a band at
162bp, meaning it was genetically modified. All tubulin samples of food and plant products,
wells 2, 4, 10, and 12, had a band at 187bp, indicating that tubulin was found in all products.
Well 11, Food Product #1 35S, had a band at 162bp, signifying it was genetically modified.
Oppositely, Well 13, Food Product #2 35S, did not have a band at 162bp and is therefore not
genetically modified.
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Well
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Content of Well
Ladder
Wildtype Tubulin
Wildtype 35S
Knockout Tubulin
Knockout 35S
Empty
Empty
Empty
Ladder
Food Product #1 Tubulin
Food Product #1 35S
Food Product #2 Tubulin
Food Product #2 35S
Empty
Empty
Empty
Table 1: Contents of Wells of H7 Gel
Figure 1: H7 Gel
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Lane
Sample
1 Ladder
2
3
4
5
6
7
8
Wildtype (-) Tubulin Control
Wildtype (-) 35S
Roundup Ready (+) Tubulin Control
Roundup Ready (+) 35S
Empty----------------------------------------------------------Empty----------------------------------------------------------Empty-----------------------------------------------------------
9 Ladder
10 Food Product #1 Tubulin
Size of Fragment
1857bp, 1058bp,
929bp, 383 bp, 121 bp
187bp
None
187bp
162bp
1857bp, 1058bp,
929bp, 383 bp, 121 bp
187bp
Band at 162
bp?
No
No
No
No
Yes
No
No
11 Food Product #1 35S
162bp
Yes
12 Food Product #2 Tubulin
187bp
No
13 Food Product #2 35S
None
No
----------------------------------------------------------------------------
-----------------------------------------------------
14 Empty----------------------------------------------------------15 Empty----------------------------------------------------------16 Empty-----------------------------------------------------------
Table 2: Results of Base Pairs of H7 Gel of Plant and Food Product
Well Number
1
2
3
4
5
6
7
8
9
Content of Well
Marker
Wildtype Tubulin
Wildtype 35S
Roundup Ready Tubulin
Roundup Ready 35S
Food Product #1
Tubulin
Food Product #1 35S
Food Product #2
Tubulin
Food Product #2 35S
Table 3: Contents of Wells from This Experiment’s Gel
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Figure 2: Gel from This Experiment
Discussion
The purpose of this experiment was to learn PCR technique as a way of identifying
whether DNA from various food and plant sources is genetically modified.
Several controls were used in this experiment as safe guards to ensure that the experiment
was working at all stages. There was a positive and negative control. The positive control was a
known genetically modified soybean, Roundup Ready and Knockout. The negative control was a
known not genetically modified Wildtype soybean. Using both a positive and negative control
allows for an accurate conclusion that the 162 bp band only shows when the sample is
genetically modified. To check that each sample properly went through the PCR process, tubulin
was identified in each sample, in addition to the 35S. Tubulin is a protein that is in living cells,
and therefore guaranteed to be seen in all samples. All the tubulin samples had a band at 187bp,
which signifies that all the samples had correctly gone through the PCR process and the
markings of the 35s samples can be deemed accurate. Another control of sorts is the ladder
placed in Wells 1 and 9. This ladder is used to tell where the bp marking are to aid in the
accurate reading of a particular band’s bp. In this experiment, the ladder had five band locations:
1857bp, 1058bp, 929bp, 383bp, and 121bp. Four of the bands can distinctly be identified in
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Figure 3. The first band at 121 bp is very faint. In Figure 2, H7 gel, not from this experiment, did
not have the exact same ladder markings. Mostly like the latter band locations, 1857bp and
1058bp may have been excluded from the ladder of H7 gel, as it was clearly known where the
band would be located to signify the presence of tubulin or genetic modification, which were
bands at smaller bp locations.
In this experiment, Gluten- Free Corn Chex cereal was used for Food Product #1 and a
Quaker Rice Cake was used for Food Product #2. The Chex cereal, being made from corn, is
genetically modified because all corn is genetically modified. Had the PCR process been
successfully, most likely Well 7, containing the Chex sample, would have had a band at 162bp.
As for the rice cake in Well 9, no definitive conclusion can be drawn. It is unknown if the
product was genetically modified. Such a conclusion can only be drawn by observing the
location of the band made in the gel.
Food Product #1 of the H7 gel was genetically modified. This can be concluded by the
marking of a band at 162bp in Well 11. It is unknown what Food Products #1 and #2 actually
are. Food Product #2 of the H7 gel was not genetically modified. This can be concluded by
absence of a band in Well 13.
Primer dimers are a byproduct of PCR. They are primers that did not attach to DNA, but
rather formed a sequence of primers. (Brownie, et al. 1997) If primers had been a byproduct of
this experiment or the H7 gel, then there would be a faint band at 50bp. There were no primer
dimer byproducts in either Figure 1 or 2.
RNA is the largest component that can be isolated form plant tissue. If RNA has been
present in the gel, it would make a band around the 121bp mark. To prevent RNA from
appearing in the gel, TE/RNase was added to help dissolve the nucleic acid pellet. TE/RNase
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eliminates the RNA, leaving only the DNA. Because this prevention measure was taken, RNA
did not appear in either gel. This can be seen in Figures 1 and 2 because there are no bands
around the 121bp mark.
From this lab, it can be concluded that PCR technique can be used to identify genetically
modified products. There were errors in this lab, but they are undeterminable as the results from
this particular experiment were never able to be gathered. Because the marker appeared in the
gel, that eliminates the possibility of there being an error with the gel. Because none of the
samples appeared, even faintly, this indicates there was an error in the PCR process. The PCR
process has three steps: denature, anneal, and extend. These happen during the heating process.
Because all groups in the lab had problems with bands appearing in the gel, it is likely that
something occurred during the thermal cycle where all samples where adjusted the same way for
the same time. Another possible error is bad products, in terms of additives and buffers. To
explore this conclusion further, this experiment should be repeated with a successful PCR
process so that the exact results from this experiment can be observed and analyzed.
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References
Janine Brownie, Susan Shawcross, Jane Theaker, David Whitcombe, Richard Ferrie, Clive
Newton, and Stephen Little. “The elimination of primer-dimer accumulation in PCR.” Nucleic
Acids Research 25.16 (1997): n. pag. 15 May 1997. Web. 14 Oct 2013.
< http://nar.oxfordjournals.org/content/25/16/3235>.
Huang, H., Cheng, F., Wang, R., Zhang, D., Yang, L. “Evaluation of Four Endogenous
Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs
Detection.” PLOS One 8.9 (2013): n. pag 30 Sept 2013. Web. 14 Oct 2013.
< http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3786954/>.