Roig 11
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 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. 100mL of Edward’s buffer was added to the four tubes. A clean plastic pestle was used to grind up the food or plant product with the buffer for one minute. 900mL 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. 350mL 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 mL 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 100mL. The air pellets were left to dry with the tube caps open for ten minutes. The isopropanol evaporated. 100mL 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, 22.5mL of the 35S primer/loading dye mix was added to the four tubes. With a fresh tip, 2.5mL 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 soybean). Using a micropipette, 22.5mL of the tubulin primer/loading dye mix was added to the four tubes. With a fresh tip, 2.5mL 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 20mL 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.
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.
Well Number / Content of Well1 / Ladder
2 / Wildtype Tubulin
3 / Wildtype 35S
4 / Knockout Tubulin
5 / Knockout 35S
6 / Empty
7 / Empty
8 / Empty
9 / Ladder
10 / Food Product #1 Tubulin
11 / Food Product #1 35S
12 / Food Product #2 Tubulin
13 / Food Product #2 35S
14 / Empty
15 / Empty
16 / Empty
Table 1: Contents of Wells of H7 Gel
Figure 1: H7 Gel
Lane / Sample / Size of Fragment / Band at 162 bp?1 / Ladder / 1857bp, 1058bp, 929bp, 383 bp, 121 bp / No
2 / Wildtype (-) Tubulin Control / 187bp / No
3 / Wildtype (-) 35S / None / No
4 / Roundup Ready (+) Tubulin Control / 187bp / No
5 / Roundup Ready (+) 35S / 162bp / Yes
6 / Empty------
7 / Empty------
8 / Empty------
9 / Ladder / 1857bp, 1058bp, 929bp, 383 bp, 121 bp / No
10 / Food Product #1 Tubulin / 187bp / 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 / Content of Well1 / Marker
2 / Wildtype Tubulin
3 / Wildtype 35S
4 / Roundup Ready Tubulin
5 / Roundup Ready 35S
6 / Food Product #1 Tubulin
7 / Food Product #1 35S
8 / Food Product #2 Tubulin
9 / Food Product #2 35S
Table 3: Contents of Wells from This Experiment’s Gel
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 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 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.