Dangerous Ideas and Forbidden Knowledge, Spring 2008
Name:
Lab #6: 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
Before coming to class, it is important that you read this handout. After reading the
handout, answer the “Pre-lab” questions in posed on page 3 on a separate piece of
paper.
INTRODUCTION
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 super-bugs 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.
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
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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 singlestranded 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 an instrument called a thermocycler.
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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.)
PRELAB
On a separate piece of paper, answer the following questions. These are due at the
start of class on your lab day.
1. In this experiment, you will add a PCR “Master Mix” to each sample. What sorts
of ingredients must be in this mix to allow the PCR reaction to work? For each
ingredient you identify, be sure to specify its function in the reaction.
2. For each sample you test in lab this week, you will set two PCR reactions. One of
these reactions, labeled “Plant”, amplifies regions of DNA found in all plants.
What is the purpose of this reaction? What does it tell you about your
experiment?
3. You will also perform PCR on two samples known to contain GMO’s (tubes 5 and 6)!
Why are these two samples included in our work? What can they tell us about the
results of our experiment?
PROCEDURES
Part A: Extraction of DNA From Food and Control Samples
1. Find your screwcap tubes containing Instagene Matrix and label one “control”
and one “test”. 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- food sample provided and put it into a
clean, uncontaminated mortar.
3. Add 5 volumes of high quality distilled water for every gram of food you weighed
out. (Thus for 2 grams of sample, add 10 mls of water.)
4. Grind with the pestle for at least two minutes to form a slurry.
5. Add a second 5 volumes 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”.
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 both tubes (“control” and “test”) to mix and place them 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 maximum speed.
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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 them 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
Master Mix
DNA
1
20 ul Plant MM (green)
20 ul Non-GMO food control
DNA
2
20 ul GMO MM (red)
20 ul Non-GMO food control
DNA
3
20 ul Plant MM (green)
20 ul Test food DNA
4
20 ul GMO MM (red)
20 ul Test food DNA
5
20 ul Plant MM (green)
20 ul GMO positive control DNA
6
20 ul GMO MM (red)
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 fridge for you to analyze during your next lab session. In the meantime, read
Part 2 of this protocol carefully before coming to lab next time.
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GMO Investigator: Part 2
BACKGROUND: Recall that in part 1 we ground up food samples, extracted their DNA,
and used this DNA to do a Polymerase Chain Reaction (PCR). This PCR will copy
specific sequences of DNA that are often used when a food crop is genetically
modified. Thus if our sample contained modified food, we expect to see our reactions
work; if the sample did not contain genetically modified food, no product should be
formed.
How can I tell if my PCR worked? Unfortunately, you cannot see individual DNA
molecules with either your naked eye or our light microscopes. So we will need another
technique to allow us to determine whether or not our PCR was successful. The
technique we will use is known as DNA electrophoresis.
How does DNA electrophoresis work? In DNA
electrophoresis, a collection of DNA molecules is
placed in wells at one end of a dense matrix called a
gel. An electrical current is then conducted across
the gel. As DNA is a negatively charged molecule, it
will be drawn towards the positive pole. (Opposites
attract!) Thus our DNA will move through the gel, and
smaller fragments will travel faster, and therefore
farther, than larger fragments. This allows us to sort the
DNA fragments from each PCR based on its size.
What will I see as my gel runs? We will mix our PCR
products with a colored loading dye. This loading dye
will make it easier to see and load our samples. In addition, the dye itself is also
negatively charged; thus is will also migrate through our gel when the current is
applied. You will see this dye moving.
The DNA produced in the PCR reaction, however, is still not visible as it has no natural
color. To see these DNA molecules, we will have to apply a DNA stain, FastBlast. This
stain will bind to the DNA molecules produced by the PCR and allow us to see them. .
Part 2: DNA Electrophoresis
Note: We will work today in the same group of 4-5 students, with each group running
their own PCR products on their gel. Make sure everyone has a chance to practice
with the micropipettes and load a sample or two!
Part A: Preparing your gel.
1. Obtain a tray from the electrophoresis supply box. CAREFULLY tape both ends of
the tray as demonstrated by your instructor. Note that these gels are prone to
leaking! A little extra time now will be worth your while! Insert a comb into one of
the sets of notches at the end of your tray.
2. Obtain a flask of melted agarose from the water bath. Carefully pour the melted
agarose into your tray, up to the line on the side. Return the melted agarose to
the water bath so other groups may use it!
3. Leave your gel in a level spot until the agarose has solidified (~10-15 minutes). The
agarose will turn slightly opaque and will be solid to the touch.
4. Gently remove the comb and the tape from both ends of your tray.
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5. Place your gel in an electrophoresis box. Recall that DNA is negatively charged,
thus it will migrate towards the positive pole (red). This means that the wells in
your gel should be at the negative (black) end of the box!
6. Carefully pour 1X TBE buffer over the surface of your gel until you have filled the
chambers at either end of your gel.
Part B: Preparing your Samples.
1. Carefully retrieve your PCR samples from last week. (Remember that these tubes
have thin walls and should be handled gently.
2. Using a fresh tip each time, carefully add 5 ul of Orange G loading dye to each
sample and mix well. On these pipettes, the dial should read “0-5-0” if your
pipette is set correctly. Remember to depress the plunger only to the first
stopping point when drawing up liquids.
Part C: Loading your Gel.
When your group has reached this point, your instructor will demonstrate how to
load a gel by loading a molecular size marker on your gel. This marker contains a
set of DNA fragments of known size and can be used to determine the size of your
PCR products. After your instructor has demonstrated the process, you may
proceed.
1. Using a fresh tip each time, carefully remove 15 ul of the PCR/Loading dye mix.
(Your pipette will read “1-5-0” this time.) Place the pipette tip at the top of the
well, and slowly dispense the solution, allowing it to sink into the bottom of the
well. Note that one of the most common errors is to push the tip too far down
and poke a hole in the bottom of the gel!
2. Record which well you placed the sample in, and continue as above to load
the remaining five samples.
Part D: Running the Gel.
1. When your gel is loaded, carefully place the cover on your electrophoresis
chamber, taking care to align red with red and black with black.
2. Plug the ends of the wires into a power supply. We will run our gels at 100V for
approximately 30 minutes. At the end of 30 minutes, your instructor will turn of
the power supply.
Part E: Staining your gel.
1. Carefully remove your gel from the electrophoresis box by lifting it out on its tray.
Note that these gels are warm, slippery, and fragile at this point!
2. Slide your gel of the tray and into a large weigh boat. Label this weigh boat with
your group’s name.
3. Pour enough 100X Fast Blast solution into the weigh boat to completely cover
the surface of your gel. Allow the solution to sit for 3-5 minutes.
4. Pour the FastBlast solution into the dedicated waste beaker provided. Begin
washing the gel by filling your tray with water and shaking gently. As the water
turns blue, pour it out and replace with fresh. You will need to repeat this
washing procedure for 15 minutes or more.
5. When the bands are clearly visible, either sketch your gel, or obtain a picture (if
the camera is available) to place in your lab notebook.
6. Clean-up: As you finish lab today, make sure your electrophoresis chambers
and gel trays have been rinsed with tap water, dried, and returned to their
storage boxes. Place these boxes on the carts in the classroom. Micropipettes
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and unused pipette tips should also be returned to their boxes. Take care to
wipe down your workspace if any solutions have been spilled. The empty plastic
tubes may be thrown in the trash.
Analyzing Your Results:
Construct a data table in your notebook to clearly indicate what each lane in your gel
contained, and how you interpret your results (i.e. assign each well a “+”. “-“, or
“inconclusive” score).
POSTLAB
1. Turn in either a picture of your gel, or a copy of the photo you took during lab. Did
each of your PCRs work as expected? Why or why not?
2. Write an Abstract describing your experiment this week. An abstract is a very brief
summary of a study, and is usually included at the beginning of a scientific paper
or poster. A good abstract should contain (in this order):
• a one or two phrase or sentence description of background (Example: "Forest
fragmentation is thought to reduce species diversity.")
• the purpose of the study (Example: "The purpose of this study was to investigate
the effects of fragmentation on diversity of salamanders in Pacific Northwest
forests.");
• a summary of the methods (e.g., "We grew the bacteria in the presence and in
the absence of arabinose, and after 15 days counted how many colonies had
grown.") -- not a lot of detail, just enough to give the reader an understanding
of how the data were collected (if the readers are interested, they can always
read your Methods section for details);
• summary of results (e.g., "Numbers of herbivores was correlated with measures
of productivity in the marsh but not the estuary."); and
• summary of conclusions (e.g., "These results indicate that competition is indeed
occurring between these two species.").
• summary of the implications/importance of your results and conclusions (e.g.,
"These results suggest that competition in these circumstances may be more
common than has previously been believed.").
Remember that your study has already been completed, so stay in the past tense!
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