GMO INVESTIGATOR, PART 1
OBJECTIVES
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To review the structure and function of DNA.
Understand and perform the polymerase chain reaction (PCR)
To gain experience using the micropipettes, thermocycler, and gel electrophoresis
To explore the benefits and potential risks of genetically modified organisms (GMOs)
PREPARATION
THIS IS A COMPLICATED EXPERIMENT. TO SUCCEED, YOU MUST CAREFULLY READ THIS
HANDOUT BEFORE COMING TO LAB!
Background: A genetically modified organism is one whose DNA has been modified, usually by the
introduction of a foreign gene. Many people are opposed to genetically modified crop plants, citing
the risk of creating super-weeds (through cross-pollination with herbicide-resistant crops) or superbugs that are no longer resistant to the toxins in the pest-resistant GM crops. Another commonly
cited concern is the potential production of allergic reactions to the new proteins used to develop the
crops. Advocates for the technology, however, argue that these crops can be better for the
environment, requiring fewer toxic chemicals. In addition, they point to the ability to preserve
farmland and improve the nutritional content of food.
Regardless of your perspective, genetically modified food is now available, with corn, soy, and papaya
being some of the most common. In the US, such foods do not have to be labeled for consumers, and
foods with less than 5% genetically modified content can be labeled “GMO-free”. Europe and Asia, in
contrast, have required genetically modified foods to be labeled if they contain more than 1% GM
content.
How do you make a GM plant? The first step in this process is to identify a gene of interest. For
example, many GM crops include a gene from a soil bacterium, Bacillus thuringiensis (Bt). This gene
produces a protein that is toxic to corn borers, a common agricultural pest.
Once the gene has been identified, scientists clone the gene, or make identical copies of it. This cloned
gene is then inserted into the target plant through one of several methods including the use of a
naturally occurring bacterium that inserts its DNA into a host plant’s genome. Plant cells may also be
induced to take up foreign DNA using an electrical current (electroporation) or by physically shooting
gold particles covered in DNA into the cells (biobalistics). Once the foreign DNA is inserted, the
plants are carefully bred to ensure that the desired traits are correctly passed on. (For more
information, please see Chapter 20 in your textbook.)
How do we know if a food has been genetically modified? In the absence of effective tracking
and labeling, GM foods can be identified experimentally by one of two methods. The first, an ELISA,
uses antibodies to detect the presence of specific proteins. This method, however, can only test fresh
produce and must be individualized according to the type of crop.
The second available test utilizes the polymerase chain reaction (PCR) to test for sequences of DNA
that have been inserted into a GM plant. As DNA is a much more stable molecule, fragments can be
isolated from highly processed foods. We will use this technique to screen for GM foods in lab this
week. Your group may choose to work either with a sample provided in lab, or with one you’ve
brought from home. If you choose to bring your own sample, consider selecting fresh corn, papaya,
corn bread mix, corn meal, soy flour, soy burgers, or other similar products.
What is PCR? In 1983, Kary Mullis at Cetus Corporation developed the molecular biology technique
known as the polymerase chain reaction (PCR). PCR revolutionized genetic research, allowing
scientists to easily amplify short specific regions of DNA for a variety of purposes including gene
mapping, cloning, DNA sequencing, and gene detection.
The objective of any PCR is to produce a large amount of DNA in a test tube starting from only a
trace amount. A researcher can take trace amounts of genomic DNA from a drop of blood, a single
hair follicle, or a processed food sample and make enough to study. Prior to PCR, this would have
been impossible! This dramatic amplification is possible because of the structure of DNA, and the way
in which cells naturally copy their own DNA.
DNA in our cells exists as a double-stranded molecule. These two strands, or sequences of bases,
bind to one another in a very specific, predictable fashion. Specifically, A’s will only pair with T’s, and
C’s will only pair with G’s. Thus if you know the sequence of one strand of DNA, you can accurately
predict the sequence of the other.
Both DNA replication and PCR take advantage of this
predictability. In your cells, one strand of DNA is used as a
template to copy the sequence of your DNA from every time
a cell divides. PCR does essentially the same process, using
one strand of your DNA as a template to produce copies of
its sequence.
PCR is conducted in three steps: 1) Denature the
template DNA, 2) Allow the primers to anneal, and 3)
Extend (copy) the template DNA. In the first step, the
template DNA is heated up to break the hydrogen bonds
holding the two strands together. This allows each strand
to serve as a template for generating copies of the DNA. In
the second step, the temperature is reduced to allow the
primes to anneal, or bind, at their complimentary sequence
on the template. (Primers are short, specific pieces of
single-stranded DNA that provide a starting point for the
enzyme that will do the ‘copying’.) In the third step, the
temperature is raised again to allow the enzyme to bind at
the primer and add bases to the growing DNA molecule.
These three steps are repeated between 20 and 40 times in a special instrument called a
thermocycler.
The power of this process is that it results in exponential growth. After the first round of copying,
a single DNA molecule will have produced two identical copies. These two copies will generate four
molecules in the next round. Those four molecules will create eight, and so on. Thus in 30 cycles we
generate literally millions of copies of DNA from each template molecule! We will be able to visualize
these millions of copies using a process called DNA electrophoresis, and thus determine whether or
not our sample contained genetic
modifications. (The process of
electrophoresis is discussed in Part 2 of
this lab handout.)
PROCEDURE
Part A: Extraction of DNA From
Food Samples
1. Find your screwcap tubes
containing Instagene Matrix and
label one “control” and one
“test” (mark tube, not cap).
Include your initials on tubes.
These are the tubes you will
place the DNA from your food samples in. (The Instagene matrix will bind any ions released
from your sample as you boil it that might otherwise interfere with your PCR.)
2. Weigh out 0.5-2.0 grams of the GM-free food sample provided and put it into the mortar.
3. Add 5 ml of distilled water for every gram of food you weighed out. (Thus for 2 grams of
sample, add 10 ml of water.)
4. Grind with the pestle for at least two minutes to form a slurry.
5. Add a second 5 ml of water to your slurry and grind further until the mixture is smooth enough
to pipette.
6. Use a disposable pipette to place 50 ul of ground slurry into the labeled screwcap tube labeled
“Control” (50 ul will be up to the first bump in the tip of the pipet).
7. Clean out your mortar and pestle as indicated by your instructor. Repeat steps 2-7 using the
food source you are testing. Place this sample in the screwcap tube you labeled “test”.
8. Shake or flick the tubes to mix and place it in a 95 C waterbath for 5 minutes.
9. Place your tubes in a centrifuge in a balanced conformation (equal number of tubes on each
side) and spin for 5 minutes at #4 speed.
10. When the centrifuge has stopped spinning, carefully remove your tubes and place them in a
rack, taking care to avoid shaking. Your extracted DNA is ready!
Part B: Setting up the PCR
Reaction
1. Obtain 6 PCR tubes, and label
caps carefully with your group’s
letter and a number. Take care
when handling these tubes as
they are delicate and crush
easily! The numbers on your
tubes should correspond to the
following tube contents:
Tube
Number
1
2
3
4
5
6
Master Mix
20 ul Plant MM (green)
20 ul GMO MM (red)
20 ul Plant MM (green)
20 ul GMO MM (red)
20 ul Plant MM (green)
20 ul GMO MM (red)
DNA
20 ul Non-GMO food control DNA
20 ul Non-GMO food control DNA
20 ul Test food DNA
20 ul Test food DNA
20 ul GMO positive control DNA
20 ul GMO positive control DNA
2. Obtain a Styrofoam cup with ice. Place each tube in an adaptor, and each adaptor into the ice to
cool.
3. Referring to the table in Step 1, and using a fresh tip for each addition, add 20 ul of the
indicated master mix to each PCR tube. Cap each tube to prevent contamination.
4. Again referring to the same table, and using a fresh tip for each tube, add 20 ul of the
indicated DNA to each tube. Be sure to avoid the InstaGene pellet at the bottom of the sample
tubes. As you add the DNA mixture to your reaction tube, pipette gently up and down to mix.
5. Be sure your tubes are tightly capped and place them in the thermocycler.
Next Steps: The thermocycler will heat and cool your samples 40 times, making millions of copies
of any regions of DNA that match the primers. This process will take several hours. When it has
finished, your instructor will place the samples in the refrigerator for you to analyze during your
next lab session. Before the next lab, answer the prelab questions for Part 2 and read Part 2 of
this protocol carefully before coming to lab next time.