Testing and Gel Electrophoresis Kacie Gresenz Lab Sect.

 

 

 

 

 

 

Testing
for GMO’s using Polymerase Chain Reaction (PCR) and Gel Electrophoresis

 

Kacie
Gresenz

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Lab
Sect. 26 – Th. 6:00pm – 8:50pm

 

 

 

 

 

 

 

Abstract

GMO’s
are all around us and there are specific methods that can be utilized to help
identify these engineered foods. The Polymerase Chain Reaction (PCR) is a fast
technique used by scientists and other professionals to amplify specific
sequences of DNA.  Following the PCR
reaction, the products can then be separated and further analyzed by using the
Agarose Gel Electrophoresis method. These methods combined are commonly used to
detect specific genes that are used to chemically engineer crops.  In this lab, we tested two corn(maize) based
products for the presence of a GMO indicating gene through the PCR and Agarose
Gel Electrophoresis methods. We concluded that our two samples were in fact
GMO’s.

 

Introduction

What
is a GMO? GMO stands for Genetically Modified Organism; Since the 1980’s, many
crops have been transformed into GMO’s in order to protect them against insects
and to reduce the overall amount of chemical pesticides sprayed on them.  Common GMO crops include: soybeans, canola,
potatoes, corn(maize), tobacco, and cotton (Bawa et al., 2012). The products we
used were those of a corn(maize) origin. How do you transform a crop into a
GMO? There are many different genes that scientists have used to genetically
transform crops into GMO’s, but the specific gene we tested for in our corn(maize)
products was cry1A.  Cry1A comes from the bacteria Bacillus Thuringiensis (Bt). 
The cry1A gene encodes for the activation of a specific Bt endotoxin
that produces a protein that is toxic to moths and butterflies.  A toxin is a poisonous substance that is
produced by living cells as well as bacteria. 
An Endotoxin is composed of lipids that are a part of a cell’s membrane;
when the bacteria is damaged the endotoxin is liberated.  Genetically modifying crops could give them
resistance to pests, herbicides, or certain environmental conditions; it could
add nutritional quality to the crop; or even give the crop tolerance to
ripening so it can last longer after being picked (Wunderlich et al., 2015).  The products we tested in our experiment came
from a corn(maize) origin.  Noting that
roughly ¾ of the processed food in the United States is genetically modified,
and that the products we tested (Yellow Corn meal and Corn Chex) were processed
foods, we as a group hypothesized that our products were indeed GMO’s.

 

How
can you find out which products are GMO’s and which products are not?  There are many processes that can be utilized
to test for GMO’s, but a fast, commonly used method by scientists and
researchers is the PCR.  PCR stands for
Polymerase Chain Reaction, it is also known as a Multiplex PCR; PCR reactions
amplify a specific base pair DNA sequence from a large group of DNA (Garibyan
et al., 2013).  What are the components
of a PCR? To perform an effective PCR reaction the assay requires a DNA
template, primers, nucleotides, DNA Polymerase, a cofactor, and a buffer.  The DNA template is the original DNA strands;
the template gives the scientist/researcher performing the experiment a
reference to compare their products to at the end of the reaction.  The primers specify the beginning and the end
of the target DNA that is to be amplified during the reaction.  The primers are small strands that act as complementary
strands to the targeted DNA strand that is going to be amplified during the
reaction.  In our experiment, we used two
different primers for each gene, a forward and a reverse primer. One primer is
used to amplify the invertase(ivr1)
gene that indicates our products are of a corn(maize) origin, the other primer
is used to possibly amplify the cry1A
gene that may or may not be present in our corn(maize) products.  Nucleotides are the building blocks of DNA;
these building blocks are called bases and there are four that are in DNA:
Adenine, Guanine, Thymine, Cytosine.  DNA
Polymerase is the enzyme that binds the nucleotides together which inevitably
form the PCR Product (Garibyan et al., 2013). 
The cofactor is used to regulate the enzyme activity, in our experiment
the cofactor used was MgCl2.  The buffer
is used to regulate the reaction to make sure it stays within its optimal pH
and temperature range.  How does a PCR
work?  The components necessary for a PCR
and your sample’s DNA extract (in this case the DNA from our corn products) are
placed into a test tube and mixed together by swirling it around.  Then the tubes are placed into a Thermocycler
– which is a machine that is used to make a bunch of copies of the DNA.  The thermocycler raises the temperature of
your samples for a given amount of time; which causes denaturation of the
proteins/enzymes/primers inside the tube (denaturation means that the
proteins/enzymes/primers will lose function and shape). Then, the machine
lowers the temperature of the samples, which allows the
proteins/enzymes/primers to do their job again (which is to replicate/amplify
the DNA of your sample).  The machine
does this cycle forty-five times (about 45 minutes), which results in extreme
amplification of your targeted DNA.  This
ultimately results into a finished PCR product that you will then use in the
next step which is the analysis of your PCR product that is easiest done through
the Agarose Gel Electrophoresis method (Garibyan et al., 2013).

What
is Agarose Gel Electrophoresis?  Gel
Electrophoresis is a method that separates the molecules of your PCR product by
charge and size by running them through an agarose gel that is charged and
porous (Stellwagen et al., 2009).   This
process is commonly done using an agarose gel which is exactly what we did when
running our gel.  How does Gel
Electrophoresis work? First you have to set a cast of the agarose gel – this is
so you can load your samples in later so they can be processed through the gel.
Then you put the mold into the electrophoresis chamber and pour a layer of
buffer over your gel.  Next, you load
your samples, DNA ladder, and negative control. 
A Negative Control is a sample in which you should see no result, a
Positive Control is a sample where you should see the result you are
expecting.  After your samples are
loaded, you plug in the electrodes and turn on the power. Remember: DNA has a slight negative charge.  Small molecules will move
through the gel faster while the bigger molecules will have a hard time moving
through the pores and will thus move slower. 
As the molecules move through the gel, bands are produced.  After running the gel, it will need to be
placed into a staining bucket/container containing ethidium bromide for 10-15
minutes so that the bands will show up distinct in the gel images.  You will then see how your samples are compared
to the negative control and the DNA ladder. 
This will then allow you to tell if your sample contains the cry1A gene indicating that it is in fact
a GMO (Stellwagen et al., 2009).

Methods

The
first part of our experiment to test for GMO’s, was to perform the PCR
reaction.  First, we needed to prepare
our corn(maize) samples by measuring out 18-20 mg of our designated corn
product.  After weighing our samples, we
then transferred our ground corn products into 1.5 mL microcentrifuge
tubes.  To the tubes we added 100 mL
(microliters) of the Extraction Solution (contains detergent that will break
open cell membranes and nuclei), and then mixed gently by swirling the tube
around so the ground corn product became hydrated.  Then we incubated out samples in a 95°C bath
for about 20 minutes.  After incubating,
we added 100mL of Dilution Solution to the tubes and vortexed them
to help it mix thoroughly.  Then, we micro
centrifuged the tubes for 1 minute on high speed until a pellet of solid
material formed at the bottom. After centrifugation, we transferred the
supernatant (liquid left in the tube containing the sample’s DNA) of each tube
into a new tube and stored it on ice. 
Ready to start the PCR reaction, we placed three 0.5mL tubes containing
the REDExtractReady Mix (which contains all the key components to an effective
PCR reaction – DNA polymerase, buffer, cofactor, nucleotides, and marker dye)
on ice along with our supernatants (liquid containing DNA).  Then we labeled two new 0.5 mL PCR tubes
indicating which sample would be in each tube (B & D), and using a P-20 micropipette
we then added: 4mL of Dilution/Extraction Solution
to a third, separate 0.5mL tube labeled “NC” for our Negative Control, 4mL of
the supernatant of each maize sample to its designated PCR tube, 10mL of
REDExtractReady Mix to all three PCR tubes, and lastly, 6mL of
primer mix (containing the forward and reverse primers for cry1A and ivr) into all three PCR tubes.  Because the Negative Control well did not
contain any DNA Extract, we should not see an effect in this lane.  Finally, after getting all the necessary
components into each PCR tube, we then placed the tubes inside the
thermocycler.  Our instructor started the
PCR thermocycler and then once finished stored them at -20°C.

The second part of our experiment
was to perform the Agarose Gel Electrophoresis to help analyze our PCR
products.  First, we prepared the gel
tray inside the casting mold; we then placed a comb at one end of the gel tray
and poured the 2.5% Agarose liquid into the mold.  The comb we placed provided wells for us to
insert our samples into the gel later on after the gel is set.  The gel took about 8 minutes to set; after it
was set we removed the comb and carefully slid the gel out of the molding tray
and into the electrophoresis unit.  Next,
we poured about 250 mL of electrophoresis buffer over the gel so the sides of
the unit were filled and a thin layer was produced over the top of the
gel.  With our gel finally ready we then
loaded our samples into the wells at the edge of the gel where the comb had
previously been. Into the first well we loaded the 20m of
the DNA ladder (which served as the reference point for our samples), the
second well was left blank, into the third well we loaded 20mL of
our Sample B, well four left blank, we made an error in well five trying to
load 20mL of our sample (we poked through the gel, leaking our
sample) so we moved the remainder of our Sample D into well seven, leaving well
six blank, and in our last well (eight) we loaded 20mL of
our Negative Control.  After loaded all
of our samples we closed the lid to the electrophoresis unit, plugged in the
electrodes (black-negative in the end by the wells, red-positive end on the
other side), and then turned on the power. 
Our starting voltage was at 80V, after 10 minutes we increased the
voltage to 120V.  Note: you should see little bubbles at the positive end of the gel –
indicates the unit is working correctly. 
After 45 minutes, we turned off our electrophoresis unit, unplugged
it and lifted our gel tray out of our unit and into a container containing
ethidium bromide; We stained our gel for about 15 minutes.  Finally, our instructor then took our gel and
placed it into a machine that took images that displayed our gel’s distinct
bands from our samples, the DNA ladder, and our negative control.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Results

After performing the PCR reaction
and the Agarose Gel Electrophoresis we concluded that our samples (Yellow Corn
Meal and Corn Chex) were in fact GMO’s. In our Agarose Gel Electrophoresis
image (Figure 1) in comparison to
our DNA ladder, the gel indicates that both the invertase(ivr1) band, and the cry1A
band are present in both samples.  Due to
our error, early on while loading our Sample D, the bands indicating that
sample are faded, but none the less still indicate the presence of the invertase(ivr1) and the cry1A genes.  Some
limitations that possibly led to error here include: pipetting skills and
contamination.  Figure 1 also displays our samples towards the positive end of the
gel, indicating that our samples particles were small enough to move fast
through the porous gel and toward the positive electrode indicating it was in
fact the (slightly negative) DNA Extract of our samples moving throughout the
gel. 

The presence of the cry1A band in both of our samples
indicate that our corn(maize) products were in fact GMO’s.

 

 

 

 

 

 

Figure 1 – with labels

 

Figure 1 – Agarose Gel Electrophoresis
Image – ivr1 and cry1A bands illuminated indicating
the corn(maize) products are genetically modified.

 

 

Discussion

The presence of the cry1A gene in both of our sample lanes
indicate that our samples were in fact genetically modified.  The results we received support our original
hypothesis – our corn(maize) samples are indeed GMO’s.  PCR procedures can be used in many different
ways and in many different types of procedures. 
PCR reactions are a common technique used in the Human Genome Project
(HGP).  PCR’s can also be used in other
processes such as DNA fingerprinting, detection of bacteria or other foreign
bodies, and diagnosis of disorders (NIH et al., 2015).  Not only are PCR’s and Agarose Gel
Electrophoresis’ used in the Human Genome Project, they are also used by the
FDA to detect the presence of GMO’s in foods (Gacheta et al., 2000).   PCR’s
and Agarose Gel Electrophoresis procedures are easy, quick methods that are
labor intensive but display clear results. 
PCR’s and Agarose Gel Electrophoresis can be used in many versatile ways
and in many different fields.  After
doing this lab I understand why these two methods are procedures of choice
chosen often by scientists, researchers, and other science professionals.