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PCR Detection of GMOs 1
PCR Detection of GMOs (Genetically Modified Organisms)
Ashley Pan
Shiwei Sun, Darrell Harvey, Nader Mehregani
Biology 210A
San Diego Mesa College
5/13/15
PCR Detection of GMOs 2
Abstract (5 points):
To find if an organism is genetically modified, PCR testing was conducted in three
separate experiments: Isolation of genomic DNA, amplification of target sequence using PCR,
and analysis of PCR products using gel electrophoresis. The test sample, fresh corn, is predicted
to be genetically modified. If the sample is a GMO, then a 200 bp band will appear in lane four
of the gel. After conclusive testing, there was no band at 200 bp, but there was evidence of
primer dimers, which means the test sample cannot be identified as GMO or a non-GMO.
Introduction/Background/Scientific Concepts (10 points):
Genetically Modified Organisms (GMOs) are often the center of controversy in the world
of agriculture. From changing the genetic makeup of organisms unnaturally without genetic
recombination or mating, these new hybrid plants are created to withstand harsh climate changes,
pesticides, or even add nutritional value to plants. This way, farmers can grow crops without
worrying if they will survive certain seasons or not. On the other hand, some argue that these
GMOs are unknown and possibly detrimental to humans since they are chemically synthesized in
a laboratory and are not “natural.”
Since around 1996 and afterwards, GM foods have reached “67.7 million hectares in
2003” (Ag Biotech). The United States has grown the most amount of GMOs, with 63% of its
crops being genetically modified. Argentina comes second with 21%, Canada with 6%, and
Brazil with 4% of its crops classified as genetically modified.
PCR Detection of GMOs 3
Compared to the rest of the world, the United States is more lax when it comes to
mentioning what has been genetically modified and what is not. Corn, soybean, cotton, and
canola are the most commonly grown GMO plants GMO positive plants (Noga 3). Often plants
are GMOs to be its own pesticide and being able to produce its own toxin to kill off insects that
try and eat it. People have argued that allergic reactions to these new substances can create new
detrimental diseases with adverse effects. Supporters of GMOs have said that GMOs can “reduce
the stress on land” by modifying them to be grown in any kind of environment.
Two methods are used to find if a food is a GMO or a non-GMO. One method is called
enzyme-linked immunosorbent assay, or ELIZA. This method includes finding proteins that are
made by GMO crops with particular antibodies. Unfortunately, since this so specific to a certain
plant, it cannot be used to test anything but fresh produce since the proteins inside could have
been destroyed through factory processing. On the bright side, ELIZA is a simple process that
can be used in the field. This means that the process is also inexpensive.
Polymerase Chain Reaction is the second method to determine if a food is a GMO or a
non-GMO. Instead of using antibodies to determine if the food is a GMO or a non-GMO, PCR
looks at sequences of DNA. This means that this method can be used for 85% of plants as well as
some processed foods since DNA is a constant for each individual plant. Sequences such as a
stop and start are used to control DNA sequences. These promoter and terminator sequences
allow certain genes to be expressed. The 35S promoter from cauliflower mosaic virus and the
nopaline synthase (NOS) terminator from Agrobacterium tumefaciens are the two most common
regulatory sequences and will be used in this experiment to identify if the test sample is a GMO
or a non-GMO food.
PCR Detection of GMOs 4
In this lab, it is important to use a sample that is fresh produce. Even though the PCR test
can be used to find GMOs, it will produce more definite bands with the gel electrophoresis
process. Corn and wheat products are more likely GMOs because they are produced in such large
quantities. Day One of this lab will be to extract the DNA from the two samples, the certified
non-GMO and the test sample. DNA isolation is necessary because this helps the PCR react
“target and amplify a specific sequence” (Day One lecture). The non-GMO sample should be
prepared first to ensure that it stays pure from the test sample in case the test sample is a GMO.
It is important to thoroughly clean the mortar and pestle after the grinding the non-GMO sample
to ensure that each sample is contained. Keep tubes closed unless needed and make sure work
surfaces stay clean.
Grinding the samples is important to the lab to extract the cells for the experiment. To
help grind the samples, DNase- and RNase- free water is needed to help grind the sample. This
special type of water is used to prevent DNases (harmful enzymes) from degrading the extracted
DNA. These samples will then be put into a solution with InstaGene matrix. InstaGene matrix
has negatively-charged microscopic beads that attach to positive cations to stop them from
binding with enzymes. This helps the sample stay intact and prevent denaturing. After
stabilization, the samples can be denatured by boiling to neutralize the DNases.
The samples will then be removed from the water bath and then put into a centrifuge.
This is so the heavier pellet will be separated from the supernatant and will sink to the bottom of
the tube. The supernatant contains the DNA needed for Day Two while the pellet contains the
InstaGene matrix beads to stop the denaturation process.
Day Two of the lab entails the PCR amplification of the lab because it can be used for
85% of plants to test whether or not they are GMOs are non-GMOs. By identifying a specific
PCR Detection of GMOs 5
sequence of DNA and replicating or “amplifying” the sequence, the process can take a small
amount of DNA and analyze the DNA on a larger spectrum. To do this, primers are needed to
“amplify” the sequence for replication. A forward and reverse primer are used to start and stop
DNA sequences and bind on complementary DNA sequences for these primers. After, the
complementary strands are copied by DNA polymerase from reading those strands, the target
sequence can then be used to read the template DNA and “add nucleotides to the 3’ ends of the
primers” (Day 2 Lecture).
Taq DNA polymerase is used because it is a thermostable DNA polymerase that can
protect the DNA’s integrity after the double-stranded DNA is separated from heating. Specific
primers, such as CaMV promote, NOS terminator, and PSII gene are used to control which
sections are needed for the target sequence for the experiment. CaMV 35S is used in the
experiment because it is found in every plant cell while NOS terminator is used because it is
“evolved to be recognized in most plants” (Biotechnology Explorer). PS II is tested to make sure
that the sample is plant based since plants undergo photosynthesis to grow. The size of the PCR
that results is much longer than the initial sequence since the process of PCR amplifies the
sequence for better observation.
PCR has three steps: denaturing, annealing, and elongation. During denaturing, the DNA
is denatured into two single strands after being heated in a water bath of 94 degrees C. DNA is
then cooled to 59 degrees C to let the primers anneal, or bond to the DNA. The primers will bond
faster to the template DNA because they are shorter than the original template DNA. The third
step is to heat the samples to 72 degrees C which lets the DNA polymerase to thrive while
allowing the DNA polymerase to add nucleotides at the 3’ end of the primer. This elongates the
3’ strand of the DNA for a complementary copy. This one cycle is then repeated around forty
PCR Detection of GMOs 6
times with a thermal cycler (PCR machine). The aluminum block inside the machine allows the
sample to be heated and cooled to allow the reaction to happen multiple times to replicate the
DNA. That way, gel electrophoresis can be used to actually see this amplification.
The master mixes used in Day Three of the lab are used as controls. The Plant Master
Mix (PPM) has plant primers to make sure that the sample used is of plant origin. The GMO
Master Mix has GM primers in it which can be used to check if the sample truly is a GMO or
non-GMO. To test the samples, an agarose gel plate must be made. A 1.5% agarose gel is used in
this part of the experiment because it is a gel that is less permeable to the samples. This creates
resistance to the bands that are created from the electric current making them easier to see with
their definition, by separating the bands more evenly. The gel as an agent to let the samples run
through it and to “paint a picture” of what the sample is. To actually see the bands, Ethidium
Bromide (EtBr) must be mixed into the melted agarose gel mix. This is so when the gel is
stained, and when put under a UV light, the bands will be fluorescent to determine how far the
sample traveled, allowing the sample to be identified as a non-GMO or a GMO. The UV light is
needed because the EtBr is visible in only UV light. Orange G dye is added to each sample as a
marker for each sample to appear on the gel. To be able to compare each marker, a molecular
weight ruler (DNA standard) is also added to each sample along with the Orange G dye to see
whether the PCR samples are supposed to be the correct size. In this lab, the ruler has 1000, 700,
500, 200, and 100 bp.
If the test sample is genetically modified, it will give a positive result with both primers
because the test sample is both a plant and genetically modified. If the sample is not genetically
modified, then the sample should have a positive reading for the plant primer, but a negative with
the GMO primer, denoted with a band at 200 bp. Considering that the test sample is corn, it is
PCR Detection of GMOs 7
most likely genetically modified because it is produced in such large quantities and corn is used
in many products such as cornmeal or feed for animals.
Materials and Methods (5 points):
Materials Day 1:
-2 screwcap tubes with 500 uL InstaGene matrix
-DNase- and RNase- free distilled water
-Plant based food samples
-Non-GMO control sample
-Clean knife to cut larger food sample
-Balance and weigh boats
-Disposable plastic transfer pipets
-Mortar and pestle
-Marker
-Mini centrifuge
-Water bath (around 95-100 degrees C)
-Micro centrifuge
-Floating micro centrifuge tube holder
Methods Day 1:
Before starting, check to see if the InsteGene matrix in the micro test tube is thawed out
and on ice. Take two screw cap tubes and label one “non-GMO” and the other “test” with the
table number.
Testing the non-GMO:
PCR Detection of GMOs 8
Weigh out 0.5g of the GMO-free oatmeal as the “certified non-GMO” control on a
weighing boat. Pour the oatmeal into a clean mortar. With a disposable plastic pipet, add 2.5 mL
DNAase- and RNase- free water to the mortar. Grind the sample with the DNAse- and RNase-
free water until a “slurry” is made. Add an extra 5 mL of the DNAse- and RNase- free water to
the mortar and again mix well until the mixture can be pipetted. Add 50 uL of the slurry to the
screwcap tube labeled “non-GMO” with the same transfer pipet in the last step. Cap the tube and
shake well.
Testing the Sample:
From the previous sample, make sure to clean and dry the mortar well before starting this
part of the experiment. Weigh out 0.5g of the test food on a weighing boat. Transfer sample to a
clean mortar. Using a clean transfer pipet, add 2.5 mL of DNase- and RNase- free water to the
mortar. Grind with a pestle until a “slurry” forms. Add another 5 mL of DNase- and RNase- free
water to the mortar until the mixture can be pipetted. Add 50 uL of the slurry to the other
screwcap container with the InsteGene matrix labeled “test” with the same transfer pipet in the
last step. Cap the tube and shake well. Wash the mortar and pestle with 10% bleach to disinfect
it.
Processing Both Samples:
Place the two screwcap tubes (the non-GMO and the test tubes) into a floating rack and
gently place into a water bath set at 95 degrees C for five minutes. Remove the rack with the test
tubes from the water bath after five minutes and then place into a balanced mini centrifuge and
spin for five minutes on maximum speed. Remove tubes from the centrifuge and store tubes in
the freezer or refrigerator until Day Two.
Materials Day 2:
PCR Detection of GMOs 9
-Micro centrifuge
-Ice bath
-GMO master mix (red tube on ice)
-Plant master mix (green tube on ice)
-GMO positive control DNA (clear tube on ice)
-Test food DNA (from Day 1)
-6 PCR tubes
-Sharpie
-2 (20 ul) micropipets
-2 (20ul) pipet tips
-thermal cycler
Preparations:
If not thawed already, thaw the two samples (non-GMO with the InsteGene matrix and
the test with the InsteGene matrix) from the previous day. Centrifuge the two samples for five
minutes to separate the supernatant (liquid with the genomic DNA) and the pellet (the solid
contents that settles at the bottom that is comprised of “cellular debris and Instagene matrix).
Label the six PCR tubes 1-6 with the table’s number. Add the designated DNA samples with the
designated Master Mix as denoted by the table taking care to keep the samples on ice. Add the
master mix first to each tube. Afterwards, with a fresh pipet tip each time, add the DNA as
directed by the table below:
Tube Number DNA Master Mix
PCR Detection of GMOs 10
1 20 uL non-GMO food control 20 uL Plant Master Mix
(green)
2 20 uL non-GMO food control 20 uL GMO Master mix (red)
3 20 uL test food DNA 20 uL Plant Master Mix
(green)
4 20 uL test food DNA 20 uL GMO Master Mix (red)
5 20 uL GMO positive control
DNA
20 uL Plant Master Mix
(green)
6 20 uL GMO positive control
DNA
20 uL GMO Master Mix (red)
Table courtesy of PCR Detection of GMOs Day Two “PCR amplification of target DNA sequences.”
Do not transfer any of the pellet with the InstaGene beads to the new tubs. If it is difficult
to obtain the pure supernatant, centrifuge again to separate the mixture from the supernatant from
the pellet. To aid in mixing, while pipetting the DNA into each designated Master Mix, pipet up
and down to mix the new solution thoroughly.
Thermal Cycler:
Place the samples into the thermal cycler and use the program “GMO-BioRad.”
Hot start 94 degrees C 2 minutes (1x)
Denature 94 degrees C 1 minute Repeat 3 steps
sequentially (40x)
Anneal 59 degrees C 1 minute Repeat 3 steps
sequentially (40x)
PCR Detection of GMOs 11
Elongate/ Extend 72 degrees C 2 minutes Repeat 3 steps
sequentially (40x)
Final Extension 72 degrees C 10 minutes (1x)
Hold 4 degrees C Infinite (1x)
Table courtesy of PCR Detection of GMOs Day Two “PCR amplification of target DNA sequences.”
Day 3:
-Agarose gel (1.5%) in a water bath
-EtBr (Ethidium bromide)
-Gloves
-Samples from Day 2
-About 300-350 mL running buffer (1 x TAE for agarose gel)
-1 vial Orange G loading dye
-1 vial PCR molecular weight ruler
-1 2-20 ul adjustable-volume pipet
-20 ul pipet tips
-Gel electrophoresis chamber
-Power supply
-UV light box
-UV goggles
Making the Agarose Gel Cast (for Electrophoresis):
Take care to wear gloves during this part of the experiment. Into the gel casting tray,
make sure that the well is tight so the agarose will not spill out when poured into the mold. Rest
the comb towards one end of the gel in one of the rungs making sure the comb is straight across.
PCR Detection of GMOs 12
While wearing gloves, add 5 uL of the EtBr (Ethidium bromide) to the bottle of melted agarose
(1.5%). Gently mix together. Pour the new mixture quickly and carefully into the mold, taking
care to not move the comb or to create air bubbles. If there are air bubbles, take a clean pipet tip
and drag the small bubbles to the longer edges of the gel. Let the gel solidify at room
temperature. When the gel is solid, carefully remove from the mold and slide into the
electrophoresis chamber. Fill the chamber with the 1X TAE buffer until the gel is covered and
the lid can still be sealed.
Loading the Samples:
With a fresh tip each time, add 10 uL Orange G loading dye to each sample. Mix well by
pipetting up and down with the same tip used to transfer the dye. Again with a fresh tip each
time, put 20 uL PCR molecular mass ruler to each tube. Separately add 20 uL of each sample
into the wells in the agarose gel made by the comb. Starting from the left, add the respected
samples to the gel taking care to fully submerge the pipet tip into each well and to not pierce the
gel with the pipet tip or to pull up too fast while loading each well to prevent contamination:
Lane Sample Load Volume
1 Sample 1: Non-GMO food
control with plant primers
20 uL
2 Sample 2: Non-GMO food
control with GMO primers
20 uL
3 Sample 3: Test food with
plant primers
20 uL
4 Sample 4: Test food with
GMO primers
20 uL
PCR Detection of GMOs 13
5 Sample 5: GMO positive
DNA with plant primers
20 uL
6 Sample 6: GMO positive
DNA with GMO primers
20 uL
7 PCR molecular weight ruler
(MWR)
20 uL
8 EMPTY 20 uL
Table courtesy of PCR Detection of GMOs Day Three “Gel Electrophoresis of PCR products.”
Gel Electrophoresis:
Set up the gel electrophoresis chamber with the corresponding colors to the machine. Run
the agarose gel at 125v for at least 30 minutes. Do not let the orange dye bleed off of the gel.
Gel Analysis:
Put on UV goggles and disposable gloves. Carefully slide the agarose gel onto the UV
light box. Turn on the light box and observe the patterns.
Results (10 points):
PCR Detection of GMOs 14
Lane Sample # bands Band sizes (bp)
1 Sample 1: Non-GMO
food control with
plant primers
1 450
2 Sample 2: Non-GMO
food control with
GMO primers
0 --------------
3 Sample 3: Test food
with plant primers
1 450
4 Sample 4: Test food
with GMO primers
? ?
5 Sample 5: GMO
positive DNA with
plant primers
1 450
6 Sample 6: GMO
positive DNA with
GMO primers
1 200
7 PCR molecular
weight ruler (MWR)
5 1000
700
500
200
100
Table courtesy of PCR Detection of GMOs Day Three “Gel Electrophoresis of PCR products.”
PCR Detection of GMOs 15
Discussion/Conclusions (10 points):
The experiment was conducted to find if the test sample was a GMO or a non-GMO
food. Using a fresh plant sample, it is easier to find if the food is a GMO or a non-GMO because
there is less processing done to it. DNA is extracted from it by manual grinding. This released
DNA is then elongated by replicated so that the sequences can be seen with the gel
electrophoresis process. The gel seemed to be a smudged with the bands. This could be because
of the evidence of primer dimers.
Primer-dimer formation “is an undesirable situation that leads to the inhibition of target
DNA amplification” (Dora 765). This can “become competitive enough to suppress the desired
product formation” (765). Primer dimers appear in gel electrophoresis. This can distort the
results of the gel plate because it makes the results harder to see. Changing the temperatures for
the water bath or the voltage for the current during the gel electrophoresis could help eliminate
the presence of primer dimers. Primer dimers mean that the results cannot be conclusive and
cannot be used to see if a sample is a GMO or a non-GMO.
The first lane was “Sample 1: Non-GMO food control with plant primers.” There was
one band in this lane at the 450 bp mark. This makes sense because the sample tested was a plant
and at around 455 bp, there should be a band. There should not be any other bands in this lane
because this was a pure sample, or a control to compare the test sample and the other control
with.
The second lane was “Sample 2: Non-GMO food control with GMO primers.” There
were no bands in this lane. This result makes sense because the control, or the non-GMO food
should have no GMO products in it, making the GMO primer useless. There should have been no
other bands in this section because this lane was just to confirm that the non-GMO food provided
PCR Detection of GMOs 16
as a control was indeed a non-GMO food. If the food was a GMO, then there would be a band in
this lane.
The third lane was “Sample 3: Test food with plant primers”. There was one band in this
lane at around 450 bp. This result makes sense because the test sample was indeed of plant
origin. This band was also very apparent which helps confirm that the DNA replication and
amplification was successful to allow the band to appear.
The fourth lane was “Sample 4: Test food with GMO primers.” It is unknown whether or
not there was a band in this lane because the area seems to be smudged. This means there was no
definite band and no way to see to confirm if the test food had GMO primers in it. Without
knowing if the food had GMO primers, there is no way to tell if the test sample was a GMO or a
non-GMO.
The fifth lane was “Sample 5: GMO positive DNA with plant primers.” There was one
band in this lane at the 450 bp mark. This should have happened because the GMO positive
should have been of plant based origin. There should not have been any other bands in this lane
because the only thing that was being tested in this lane was if the food was a plant or not.
The sixth lane was “Sample 6: GMO positive DNA with GMO primers.” This lane had
one band. This lane should have had one band at the 200 bp mark. This makes sense because the
200 bp band means there are GMO primers in the mixture. And since this sample had GMO
positive DNA, it should have had a band here.
The seventh lane was the “PCR molecular weight ruler (MWR)” This lane had five
bands. These bands should have appeared at the 1000, 700, 500, 200, and 100 marks. The gel
plate had these five bands to show that this standard is a valid measuring tool to look at the bands
in the agarose gel.
PCR Detection of GMOs 17
Primer dimers were observed on the gel plate because it there were multiple shadows in
lane four. This means that there were some sources of error in the experiment. One error that
could have occurred could be from the pipet tips being contaminated. With many pipet tips being
used, it could have been possible to mix up the samples. Another mix up that could have
occurred would have been from not labeling the right screw tubes with the right names. This
could have mean getting a result that could have been misinterpreted as another and leading to
false conclusions.
While grinding the samples, not washing the mortar and pestle thoroughly between each
sample (the non-GMO and the GMO positive), the non-GMO could have been contaminated
with the GMO positive or vice versa. Also, not grinding the samples enough could have been
another possible error because it would have not allowed the DNA to be completely extracted for
the second day. Not using DNase- and RNase- free water also could attribute to errors. Using
regular water, or even distilled water could have led to the denaturing of the DNA too early
which would have been detrimental to the PCR process.
During the extraction of the DNA from the supernatant, some of the pellet (with the
InstaGene matrix) could have been pipetted up and transferred. This would have hurt the
experiment because the beads in the complex would have removed certain cations that Taq
polymerase needs to work.
Creating the agarose gel could have also had many sources of error. If the gel was not
mixed well enough with the EtBr, then the gel would have had glowing splotchy places which
would not reflect the bands well. Bubbles from pouring the gel in the mold would have hindered
the passing of the current through the gel. If the gel was not immersed completely in the buffer,
PCR Detection of GMOs 18
this would have also not allowed the current to completely pass through the gel for a clean band
at the end.
Pipetting the samples into the gel could have had some errors like missing the well and
injecting the sample into the buffer. This would contaminate all of the other wells and samples. If
the pipet tip was stabbed into the gel and pierced the gel, this would also make the sample bleed
into the other wells leading to more contamination between the standards and the samples.
If the samples were not completely centrifuged after each extraction and mixing, then this
could have contaminated the samples with each other, giving the samples a false positive where
there should not have been. This process could have been easier if there could have been slightly
larger quantities or clear screw tubes so it would be easier to see the supernatant separate from
the pellet at the bottom.
There was one point in the Orange G dye process where a pipet was double dipped into
the original tube. This was remedied by stopping all of the additions and getting a new sample
from the instructor to make sure that no samples could have been contaminated.
For people who are intent on living healthy, knowing the process of PCR, or even ELIZA
could test which foods are GMO positive. PCR testing could be used to isolate DNA sequences
to see if certain foods have diseases or could have harmful effects on humans. In the future, tests
like these could be more easily accessible with more immediate results with something like test
strips.
This experiment was inconclusive because there was no definite band in lane four. This
means that even though the other standards were definite, there was no way to identify if the test
sample, fresh corn, was a GMO or a non-GMO. The hypothesis for the experiment is proven
false because the corn could not be determined as a GMO.
PCR Detection of GMOs 19
Stance on GMOs:
I believe that GMOs are relatively harmful things. I dislike how in the United States the
policies on stating on if a food is a GMO, it does not have to be listed depending on the
percentage that has been actually genetically modified. Companies such as Monsanto profit on
genetically modifying crops, and are in such control of the agriculture business, that they are
unstoppable. I believe that it should be a choice whether or not someone wants to eat a food that
has been genetically modified.
In terms of foods that have been processed that have ingredients that could have been
genetically modified, I think it is fine only because of the processing may have killed or
completely changed the composition of the food so it would not be so detrimental to my health.
PCR Detection of GMOs 20
References (5 points):
Brahmbhatt, A. (Director) (2015, April 15). PCR Detection of GMOs (Genetically Modified
Organisms). Lecture conducted from , San Diego.
Brahmbhatt, A. (Director) (2015, April 22). PCR Detection of GMOs (Genetically Modified
Organisms) . Day Two (PCR Amplification of Target DNA Sequences). Lecture conducted from
, San Diego.
Brahmbhatt, A. (Director) (2015, April 29). PCR Detection of GMOs (Genetically Modified
Organisms). Day Three (Gel Electrophoresis of PCR Products). Lecture conducted from , San
Diego.
Dora, D., Kocagöz, T., & Öner, F. (2008). Synthesis and Cloning of a Small Bacillus Pheromone
Gene (ComXRO-B-2) by Primer-Dimer Formation with PCR. Turkish Journal of Chemistry,
32(6), 8-8. Retrieved May 13, 2015, from Ebsco Discovery Services.
F. Noga, K. (2014). Fields of Change: Municipal GMO Regulation and the Federal Takings
Clause. Vermont Law Review, 39(2), 37-37. Retrieved May 13, 2015, from
http://eds.a.ebscohost.com.libraryaccess.sdmesa.edu/eds/pdfviewer/pdfviewer?sid=cf59836c-
aa9f-4fa8-8687-090dc91e7d38@sessionmgr4003&vid=0&hid=4208
Hitomi, S., Brown, K., Andrews, S., & Levi, E. (n.d.). GMO Investigator Kit- Bio Rad.
Retrieved May 13, 2015.
Rizzi, A., Sorlini, C., & Daffonchio, D. (2004). Practicality of Detection of Genetically Modified
Organisms (GMOs) in Food. AgBiotechNet, 6, 9-9. Retrieved May 13, 2015, from
http://www.researchgate.net/profile/Daniele_Daffonchio/publication/228593692_Practicality_of
PCR Detection of GMOs 21
_detection_of_genetically_modified_organisms_(GMOs)_in_food/links/0deec5159d30318b7f00
0000.pdf