Qiu 1

Determining the presence of rDNA in food sample

Catherine Qiu; Noor Faralina Zainul Fadziruddin
Submitted: October 8, 2015
INTRODUCTION
The use of GMO crops has seen a significant increase in recent years and will likely continue in
this upwards fashion. Though these are also produced for animal feed and biofuel, it is
predominantly its use in human GMO foods that the public is aware of. Whether it manifests
itself directly as a genetically modified crop or indirectly through the incorporation of a
component of a GM crop into processed food, the proliferation of recombinant DNA within our
food supply is certainly becoming common. The benefits of agricultural biotechnology are
certainly clear that is, to address concerns such as being able to feed a rapidly expanding
population with declining availability of arable land and water while at the same time dealing
with effects of global climate change (Takeda, S. et al., 2008).
In this experiment, we test an everyday food product for the presence of rDNA. This is
conducted by extracting the DNA from the sample and using pre-determined primers to amplify
either the Cauliflower Mosaic Virus 35S Promoter (CaMV 35S) and/or nopaline synthase
terminator (NOS), which are commonly used during genetic modification of crops. GMO and
non-GMO controls will be tested simultaneously to ensure reliable results. Specifically, organic
spinach purchased from Walmart was chosen to be the test food sample to confirm if the organic
certification holds up to its standard of remaining GMO-free.
METHODS
1. Extraction of DNA from food samples and setting up PCR reactions
DNA was extracted from our food sample, organic spinach, and a non-GMO food control, oats,
by initially weighing out the same amount to be ground up in distilled water to smoothness.
Extraction of samples was conducted separately to avoid contamination. Next, each of the food
slurries was combined with InstaGene Matrix to prevent the enzymatic degradation of DNA. The
sample was heat-shocked by exposing it to 95C then immediately plunging into ice. Samples
were microfuged before transferring to PCR tubes containing PCR reagents according to Table 1
(Table 1). Tubes were subjected to gene amplification in the Thermocycler.
2. Gel Electrophoresis of PCR products
A pre-prepared 3% agarose gel containing ethidium bromide was placed in a gel apparatus filled
with TAE buffer and ethidium bromide. Orange G loading dye was added to each of the six PCR
product samples before loading onto the gel according to the Gel Loading Map (Table 2). The gel
was run until the dye reached about halfway down the gel. Images of the gel were taken using
the BioDoc-It.
RESULTS
PCR products of our food sample and controls were loaded onto an agarose gel and visualized by
UV light as shown by Figure 1 (Fig. 1). Lanes 1, 3, and 5 had one band each around 455 bp, as
marked by the molecular weight ladder. Lanes 2 and 4 had no visible bands. And lane 6
contained one band around 195 bp. The band sizes were tabulated in Table 3 (Table 3). The
blurry bands in lanes 2, 4, and 6 represent the migration of loading dye within the sample.

Qiu 2

DISCUSSION
Analysis of the results shows that firstly, the non-GMO control and GMO positive control
produced expected results. Samples loaded with plant primers should all produce a band around
455 bp if it is a plant-based food sample. This was indeed the case as a single 455 bp band
appeared in lanes 1, 3, and 5. This indicates the plant primers are working and DNA from our test
food was able to be extracted. In addition, GMO primer products should have produced a 203 bp
band representing the CaMV 35S promoter only in our GMO positive control as well as our food
sample if it contained rDNA. Lane 6 with the positive control did reveal a band that, although
wasnt exactly 203 bp, was within the range approximately 195 bp. The slight deviation may
be due to unevenness of the gel or variability during the process of loading PCR products onto
the gel. Nonetheless, these results allow for the validation of our experiment so the results
obtained from the test food can be deemed reasonably reliable. It was thus concluded that our
test food, organic spinach, did not indeed contain rDNA, based on the lack of any bands in lane 4
with GMO primers. However, a caveat to this conclusion is that only primers designed to detect
the CaMV 35S promoter and NOS terminator were used in this experiment. It could very well be
that there may be rDNA within our test food that contain a different set of promoters and
terminators used during the genetic modification of the original crop. Future experiments could
examine different sequences of genes that are indicative of a GMO food.
Similar experiments could be conducted to isolate and even sequence specific genes that mark
the presence of rDNA. This would be especially useful for government agencies regulating the
labeling of foods such as those under the guidelines of organic-certified. In addition,
companies conducting quality control on their GMO seed products would be interested in
verifying whether these products truly do contain the desired recombinant genes. In conclusion,
our experiments successfully showed that our test food, organic spinach, tested negative for
rDNA content.
REFERENCES
1. Takeda, S. & Matsuoka, M. (2008). Genetic approaches to crop improvement: responding to
environmental and population changes. Nature Reviews, Genetics 9, 444.
FIGURES AND TABLES
Table 1. PCR Reaction Mixtures
Tube #
PCR Master Mix
1
Plant MM (green)
2
GMO MM (red)
3
Plant MM (green)
4
GMO MM (red)
5
Plant MM (green)
6
GMO MM (red)

DNA Sample
Non-GMO Food Control
Non-GMO Food Control
Test Food
Test Food
GMO Positive Control DNA
GMO Positive Control DNA

Qiu 3
Table 2. Gel Loading Map
Lane #
1
2
3
4
5
6
7
8

PCR product sample loaded

Non-GMO Food, Plant Primers
Non-GMO Food, GMO Primers
Test Food, Plant Primers
Test Food, GMO Primers
GMO DNA, Plant Primers
GMO DNA, GMO Primers
PCR DNA Ladder
BLANK

Figure 1. Gel with PCR product samples

MW (bp)

200

Table 3. Approximate DNA band sizes

Lane #
1
2
3
4
5
6

Estimated size of band (bp)

455
None
455
None
455
195