Jackson Wyers
MARB 301 Lab
GMO Lab Report
3/20/13
Testing foods for the presence of genetic modification by methods of Polymerase Chain
reactions and Gel Electrophoresis
Introduction - Genetic modification is a common practice in the modern agriculture
industry. This exercise is so popular because of the option to manipulate genes in one’s own
favor to produce desired traits. GMOs – genetically modified organisms – are the basis for
genetically modified crops that are commonly found today. Although favored by many, genetic
modification of foods remains a controversial topic. Many argue that these crops are not as
healthy as complete organic crops. Some also assert that organisms may evolve that could
express a resistance to commonly used pesticides. On the other hand, those in favor of genetic
modification claim that the benefits far outweigh the risks. Some positives of this practice
include larger crop yields, an increase in nutritional value, and less stress on the land that is
farmed.
Due to the controversy of GMO usage, foods are frequently and strenuously tested by
methods of the polymerase chain reaction (PCR) to see whether genes have been manipulated or
not. PCR is used in order to amplify the DNA for analysis. In order to extract DNA, chemical or
physical procedures are taken in order to induce cell lysis. In the case of this experiment,
physical grounding of the food will be used to cause the lysing. The PCR technique has three
steps that occur in the process of amplifying the DNA; denaturation is the first to occur. The
hydrogen bonds are broken by high temperatures and the DNA separates. The next step is the
annealing step in which the temperature is lowered and primers attach to the template DNA.
Finally, elongation occurs and DNA polymerase will tack on dNTP’s in order to generate a
compliment strand. For this experiment, two different primers are used. Primers specific to a
certain gene in plants was used in order to know if the DNA is successfully extracted. In this
case, DNA will be amplified whether the food is GMO or non-GMO. Secondly, a GMO primer
is used in order to identify the presence of GMO’s. The DNA will only be amplified if the food
has undergone genetic modification. Gel electrophoreses will be the final step of this experiment
in order to visualize the DNA bands and determine if the test food has been genetically modified.
Materials & Methods – For this experiment, the first step was to obtain a non-GMO
control. 500µl of InstaGene matrix was inserted into two screwcap tubes. One tube was labeled
“Non-GMO” and the other was labeled as “Test.” 1.0 gram of certified non-GMO was weighed
out and placed in a small tub. 5mL of distilled water was then added. This mixture was then
ground up with a screw cap for several minutes in order to form a slurry mix. Another 5mL of
water was added and then ground some more to create a smooth pipetteable mixture. 50µl of the
slurry was then inserted into the screwcap tube labeled “Non-GMO.” After shaking the tube and
placing it back in the tray, the tub containing the slurry and the screw cap were washed with soap
and dried for a repeat of the process. This time, a test food was chosen – Ramen Noodles in this
case – and the process for making a slurry was repeated. Afterwards, 50µl of this new slurry was
added to the tube labeled “Test.” Following this, the two tubes were placed in a 95ºC dry bath for
5 minutes. Once done, the tubes were balanced carefully and were placed into a microcentrifuge
to spin for roughly 5 minutes at maximum speed. During this time, 6 PCR tubes were labeled #16. Table 1 gives reference to the contents of the numbered tubes.
Table 1: DNA and Master Mix contents of PCR tubes #1-6
Tube Number
1
DNA
20µl non-GMO food control
Master Mix
20µl Plant master mix (green)
DNA
2
20µl non-GMO food control
20µl GMO master mix (red)
DNA
3
20µl Test food DNA
20µl Plant master mix (green)
4
20µl Test food DNA
20µl GMO master mix (red)
5
20µl GMO positive control
20µl Plant master mix (green)
DNA
6
20µl GMO positive control
20µl GMO master mix (red)
DNA
After removing the tubes from the centrifuge, a pipette was obtained and the indicated master
mix was added to the corresponding tubes while using a sterile tip each time. Green plant master
mix (PMM) was added to tubes 1, 3, and 5 while the red GMO master mix (GMM) was added to
tubes 2, 4, 6. Following the addition of the appropriate master mix, 20µl of non-GMO food
control was added to tubes 1 and 2. Test food samples of 20µl were then similarly added to tubes
3 and 4. GMO positive DNA was then added to tubes 5 and 6. Once labeling was completed, the
6 PCR tubes were placed in the thermal cycler to run for a number of hours. The next portion of
methods was initiated during the next week of lab meeting time. Because the experiment was
split into two weeks, the tubes were kept refrigerated after removal from the thermal cycler. The
next step of the procedure after placing the tubes in the thermal cycler was to conducted gel
electrophoresis. For this specific DNA, a 2.0% agarose gel was made instead for a higher
selectivity of DNA. The typical procedure for making a gel was followed by putting 1.0 grams of
Molecular Biology Grade agarose in 50 mL of 1X TAE buffer and the microwaved to dissolve.
Ethidium Bromide (EtBR) was then added to the mix. Instead of the typical 3µl of Ethidium
Bromide, 5µl was used as a substitute in hopes of making the UV pictures brighter. After the
EtBr addition, the gel was poured into the gel tray, a gel comb was inserted, and the gel mixture
was left to cool and solidify. While cooling, 10µl of OrangeG dye was added to each of the
tubes. After the gel was cool, the comb was removed and the gel tray was filled with 1X TAE
buffer. 20µl of PCR ruler was added to lane 1 of the gel. Tubes 1-6 were then added to lanes 2-7
in consecutive order of 20µl amounts. Once the lanes were loaded, the gel was run for
approximately 45 minutes at 100 volts. When completed, the gel was photographed with the
Kodak Gel Logic 112 UV imaging system and the resulting image can be seen in Figure 1.
Results – Figure 1 is the result from the
PCR and Gel Electrophoreses to test for genetically
Figure 1: This figure shows is the result of using
PCR to test for GMO's
modified organisms. By observation from right to
left, the ladder was successful however is not very
clear. Lanes 2, 3, and 4 did not even successfully
run however, they were visible. Lane 5 did not even
appear, while lanes 6 and 7 did show up.
Discussion – The purpose of this
experiment was to test a sample of Ramen Noodles
for the presence of GMOs. Observing the results, it appears that significant error(s) occurred.
The GMO positive control DNA of both plant master mix and GMO master mix seemed to have
been successful. For both of the non-GMO controls and the test food DNA with the plant master
mix, it would seem as if DNA was not amplified because there was no movement. However, lane
3 – the non-GMO with GMO master mix – should not have been amplified because there is no
presence of GMO’s in the non-GMO control. Lane 5 – the test food DNA with GMO master mix
– is not visible. This presents two possibilities; either the DNA was not successfully transferred
or the DNA was not amplified because the food did not contain GMO’s. The likely reason for
lanes 2, 3, and 4 not running is that the DNA was too “bulked” up. Whether it was large protein
substance or just foreign material in the sample, the DNA was prevented from running. Since the
ethidium bromide can be seen clearly in the three wells, it would seem that the DNA just never
left the well due to an inability to run in the highly selective 2.0% gel. Lanes 6 and 7 – the
positive GMO controls – ran successfully and were clearly visible to use for comparison.
A known error happened when inserting the OrangeG loading dye into the tubes. Instead
of adding the 10µl of dye to each tube, 20µl was added. This would mean that the overall
mixture in each tube was now more diluted than it should have been. It is possible that that is
responsible for some of the bands not being very intense however, it does not explain why some
of the DNA appeared not to work at all. Another error was noted when some of the solution in
lane 2 spilled over into lane 3. Since these two lanes were the non-GMO test lanes, this error
probably had little effect on the problem previously discussed.
A definitive answer cannot be given as to whether the tested food was GMO positive or
negative. However, with the absence of a band in lane 5 – the test food with GMO master mix –
it is not unlikely that the Ramen Noodles did not contain any GMOs. Yet, with the errors present,
this cannot be confirmed unless this experiment were to be ran again correcting the amount of
dye used and giving careful attention not to contaminate the solution.