Notes

Protocol (wash hands and wear gloves for the entire protocol):
Each team member will have to successfully adjust the pipettor and transfer a specific amount of liquid from a weigh dish
to a square of parafilm. After each member of the team has demonstrated their proficiency with the micropipettor, you
can proceed to aspects of the DNA extraction that require the micropipettor.
Note: ALWAYS process the non-GMO control before the test sample to reduce the risk of contamination.
(i) Grind non-GMO food control (Oatmeal at your bench in a covered weigh boat)
1. Find your screwcap tubes containing 500 µl of InstaGene matrix and label both with your group number
and one “non- GMO” and the other one “test”.
2. R e c o r d t h e weight __________ g of the certified non-GMO food control (oatmeal) and place in mortar.
3. Using a disposable transfer pipet, add 5 ml of distilled water from the 50 ml Falcon tube at your station for
every gram of food using the graduations on the transfer pipet. To calculate the volume of water you need, multiply
the mass in grams of the food weighed out by 5 and add that many milliliters.
Mass of Food = g x 5 = ml
4. Grind with pestle for at least 2 min until a slurry is formed.
5. Being careful not to contaminate the distilled water with slurry, add 5 volumes of water again and mix or grind further
with pestle until the slurry is smooth enough to pipet. Check with your TA before proceeding to the next step.
6. Add 50 µl of ground slurry to the screwcap tube containing 500 µl of InstaGene mat
2. Using the transfer pipet, add 5 ml of distilled water for every gram of food using the graduations on the
transfer pipet. To calculate the volume of water you need, multiply the mass in grams of the food weighed
out by 5 and add that many milliliters.
Mass of food = g x 5 = ml
3. Grind with pestle for at least 2 min until a slurry is formed.
4. Add 5 more volumes of water and mix or grind further with pestle until the slurry is smooth enough to pipet. Check
with your TA to ensure that your slurry is ground sufficiently.
5. Add 50 µl of ground slurry to the screwcap tube labeled “Test” using the 50 µl mark on a transfer pipet.
6. Recap tube and mix well.
(iii) Process Samples to Extract DNA:
1. Place non-GMO food control and test food sample tubes in 95°C heat block for 5 min.
Heat block
2. Place tubes in a centrifuge in a balanced conformation and spin for 5 min at max speed. Your TA
will confirm that the centrifuge is balanced before spinning.
3. Place tubes on ice and proceed to set up for PCR. Be very careful not to resuspend the pellet at the bottom of the
tube. The supernatant at the top of the tube contains intact extracted DNA.
Part B: Set Up PCR Reactions
In the last activity you extracted DNA from a certified non-GMO food sample and a test food sample that you are analyzing for
the presence of GMO DNA sequences. You will now prepare those two samples and a positive control (GMO-positive template
DNA) for the polymerase chain reaction (PCR).
PCR is DNA replication in a test tube. PCR allows you to amplify specific sections of DNA and make millions of copies of the target
sequence. Your experiment is to determine whether or not the DNA you extracted from food in Part 1 contains or does not contain
the target sequences of interest typically found in genetically modified (GM) foods.
What is PCR?
PCR is such a powerful tool because of its simplicity and specificity. All that is required are tiny quantities of the DNA template you
want to amplify, DNA polymerase, two DNA primers, four DNA base pair subunits (deoxyribonucleoside triphosphates of adenine,
guanine, thymine, and cytosine) and buffers.
Because PCR identifies a specific sequence of DNA and makes millions of copies of (or amplifies) that sequence, it is one of the most
useful tools of molecular biology. Scientists use PCR to obtain the large amounts of a specific sequence of DNA that are necessary
for such techniques as gene cloning, where DNA is physically moved from one genome to another. You are using the property of
PCR that allows identification of a specific sequence, namely, the ability of PCR to search out a single sequence of a few hundred
base pairs in a background of billions of base pairs. For example, the corn genome has 2.5 billion base pairs of DNA. This sequence
is then amplified so that there are millions of copies of it so that it stands out from the few copies of the original template DNA.
PCR locates specific DNA sequences using primers that are complementary to the DNA template. Primers are short strands of DNA
(usually between 6 and 30 base pairs long) called oligonucleotides. Two primers are needed to amplify a sequence of DNA, a
forward primer and a reverse primer. The two primers are designed and synthesized in the laboratory with a specific sequence of
nucleotides such that they can anneal (bind) at opposite ends of the target DNA sequence on the complementary strands of the
target DNA template. The target DNA sequence is copied by the DNA polymerase reading the complementary strand of template
DNA and adding nucleotides to the 3' ends of the primers (see fig 2). Primers give the specificity to the PCR, but they are also
necessary because DNA polymerase can only add nucleotides to double-stranded DNA.
During PCR, double-stranded DNA template is separated by heating it, then each primer binds (anneals) to its complementary
sequence on each of the separated DNA strands, and DNA polymerase elongates each primer by adding nucleotides to make a
new double-stranded DNA (see fig 2).
The DNA polymerase used in PCR must be a thermally stable enzyme because the PCR reaction is heated to 94°C, which would
destroy the biological activity of most enzymes. The most commonly used thermostable DNA polymerase is Taq DNA polymerase. This
was isolated from a thermophilic bacterium, Thermus aquaticus, which lives in high-temperature steam vents such as those in
Yellowstone National Park.
Figure. 2. A complete cycle of PCR.
PCR Step by Step
PCR has three steps, a denaturing step, an annealing step, and an elongation step. During the denaturing step, the DNA template
is heated to 94°C to separate (or denature) the double-stranded DNA molecule into two single strands. The DNA is then cooled to
59°C to allow the primers to locate and anneal (bind) to the DNA. Because the primers are so much shorter than the template DNA,
they will anneal much more quickly than the long template DNA strands at this temperature. The final step is to increase the
temperature of the PCR reaction to 72°C, which is the optimal temperature for the DNA polymerase to function. In this step the
DNA polymerase adds nucleotides (A, T, G, or a C) one at a time at the 3’ end of the primer to create a complimentary copy of the
original DNA template.
These three steps form one cycle of PCR. A complete PCR amplification undergoes multiple cycles of PCR, in this case 40 cycles.
The entire 40 cycle reaction is carried out in a test tube that has been placed in a thermal cycler or PCR machine. This is a machine
that contains an aluminum block that can be rapidly heated and cooled. The rapid heating and cooling of this thermal block is
known as thermal cycling.
Two new template strands are created from the original double-stranded template during each complete cycle of PCR. This causes
exponential growth of the number of target DNA molecules, i.e., the number of target DNA molecules doubles at each cycle; this
is why it is called a chain reaction. Therefore, after 40 cycles there will be 2
40, or over 1,100,000,000,000 times more copies
than at the beginning. Once the target DNA sequences of interest have been sufficiently amplified, they can be visualized using
gel electrophoresis. This allows researchers to determine the presence or absence of the PCR products of interest.
Part B: PCR to detect GMO sequences in food samples
For this experiment you will set up two PCR reactions for each DNA sample, which makes 6 PCR reactions in total. One PCR
reaction, using the plant master mix (PMM), is a control to determine whether or not you have successfully extracted plant DNA
from your test food. This is done by identifying a DNA sequence that is common to all plants by using primers (colored green in
the kit) that specifically amplify a section of a chloroplast gene used in the light reaction (photosystem II). Why is this necessary?
What if you do not amplify DNA using the GMO primers? Can you conclude that your test food is not GM or might it just be that
your DNA extraction was unsuccessful? If you amplify DNA using the plant primers, you can conclude that you successfully amplified
DNA, therefore whether or not you amplify DNA with your GMO primers, you will have more confidence in the validity of your
result.
The second PCR reaction you carry out will determine whether or not your DNA sample contains GM DNA sequences. This is done
by identifying DNA sequences that are common to most (~85%) of all GM plants using primers specific to these sequences. These
primers are colored red and are in the GMO master mix (GMM).
Why do you have to set up a PCR reaction with DNA from certified non-GMO food? What if some GMO-positive DNA got into
the InstaGene or master mix from a dirty pipet tip or a previous class? This DNA could be amplified in your test food PCR reaction
and give you a false result. By having a known non-GMO control that you know should not amplify the GMO target sequences
you can tell if your PCR reactions have been contaminated by GMO-positive DNA.