INSTRUCTIONDNA sampling and genotyping experiment
Teacher’s Version
Hate Brussels sprouts?Your parents may be to blame.
In 1931, a scientist named Arthur Fox spilled a
chemical called phenylthiocarbamide (PTC),
causing PTC dust to fly throughout the lab. A colleague
standing nearby immediately complained about a bitter
taste in his mouth. Arthur Fox was intrigued, since
he did not taste anything even though he received a
higher exposure to the cloud of PTC particles. To try to
understand what was going on, Arthur Fox conducted
an experiment. He asked several of his friends and
family members to taste the PTC. It turned out that
some of the people he tested were able to taste the PTC
while others did not taste anything.
In 2003, scientists linked the ability to taste PTC to
a gene named TAS2R38. This gene allows us to
taste bitter compounds that are mainly found in plants.
In this particular gene, there are 3 single-nucleotide
polymorphisms (SNPs) that can affect the ability
to taste the bitter PTC. These types of mutations
are common in our DNA and explain some of our
differences.
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This is the DNA sequence of
the TAS2R38 gene. In red,
you can see the three locations
where people can have different
nucleotides.
1 ATGTTGACTC TAACTCGCAT CCGCACTGTG TCCTATGAAG TCAGGAGTAC ATTTCTGTTC 61 ATTTCAGTCC TGGAGTTTGC AGTGGGGTTT CTGACCAATG CCTTCGTTTT CTTGGTGAAT 121 TTTTGGGATG TAGTGAAGAG GCAG G/C CACTG AGCAACAGTG ATTGTGTGCT GCTGTGTCTC 181 AGCATCAGCC GGCTTTTCCT GCATGGACTG CTGTTCCTGA GTGCTATCCA GCTTACCCAC 241 TTCCAGAAGT TGAGTGAACC ACTGAACCAC AGCTACCAAG CCATCATCAT GCTATGGATG 301 ATTGCAAACC AAGCCAACCT CTGGCTTGCT GCCTGCCTCA GCCTGCTTTA CTGCTCCAAG 361 CTCATCCGTT TCTCTCACAC CTTCCTGATC TGCTTGGCAA GCTGGGTCTC CAGGAAGATC 421 TCCCAGATGC TCCTGGGTAT TATTCTTTGC TCCTGCATCT GCACTGTCCT CTGTGTTTGG 481 TGCTTTTTTA GCAGACCTCA CTTCACAGTC ACAACTGTGC TATTCATGAA TAACAATACA 541 AGGCTCAACT GGCAGATTAA AGATCTCAAT TTATTTTATT CCTTTCTCTT CTGCTATCTG 601 TGGTCTGTGC CTCCTTTCCT ATTGTTTCTG GTTTCTTCTG GGATGCTGAC TGTCTCCCTG 661 GGAAGGCACA TGAGGACAAT GAAGGTCTAT ACCAGAAACT CTCGTGACCC CAGCCTGGAG 721 GCCCACATTA AAGCCCTCAA GTCTCTTGTC TCCTTTTTCT GCTTCTTTGT GATATCATCC 781 TGTG T/C TGCCT TCATCTCTGT GCCCCTACTG ATTCTGTGGC GCGACAAAAT AGGGGTGATG 841 GTTTGTGTTG GGATAATGGC AGCTTGTCCC TCTGGGCATG CAGCC A/G TCCT GATCTCAGGC 901 AATGCCAAGT TGAGGAGAGC TGTGATGACC ATTCTGCTCT GGGCTCAGAG CAGCCTGAAG 961 GTAAGAGCCG ACCACAAGGC AGATTCCCGG ACACTGTGCT GA
In this experiment, we will focus on one of these
locations. The nucleotide at position 145 on the
gene can be either a G or a C. People who have
a G are usually able to taste PTC, and people
who have a C are usually unable to.
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Humans are diploid, meaning they have 2 sets of chromosomes: one from the mother
and one from the father. Therefore, it is possible that you could have inherited:
The first two combinations (GG or GC) should make it possible for you to taste the PTC a little or a
lot. The third one (CC) should make you unable to taste it. About 80% of people have at least one G
and are able to taste PTC, and about 20% of people have two Cs and are not able to taste PTC.
In this experiment, you will use your own DNA to see if you are part of the 80% of people who can
taste PTC, or part of the 20% of people who are unable to.
One “G” from each of your parents
One “G” from one of your parents and one “C” from your other parent
One “C” from each of your parents
G
GG GC CC
G G C C C
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First, let’s see what your tongue has to say!Using the PTC strips provided in the kit, have students place one on their tongue and taste it.
Use this spreadsheet to record how much each student is able to taste the PTC.
Can you come up with any reasons why there would be a difference in our ability to taste (taste a lot, a little, or not at all) bitter compounds?
Do your genes agree with your tongue?
How much did you taste? A lot A little Didn’t taste anything
Name Tasted a lot Tasted a little Tasted nothing
Notes for teachers:Many toxic compounds in plants are bitter. In the early evolution of humans, the ability to taste bitter flavors may have helped us avoid toxic plants and therefore survive. However, if early humans had all detected the bitter flavors, they might have had trouble finding enough food. The mild tasters would have been more likely to eat vegetables, but might still have been able to taste the toxic ones. Some vegetables now known to be nutritious are also somewhat bitter (e.g., cabbage and broccoli). Being a mild taster or non-taster could make you more likely to eat those vegetables and get their benefits.
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For the molecular work, we are going to perform a genotyping
experiment. Here is the overview:
First, you will use a cotton swab to collect cells from the inside of your
cheek.
We will then make the DNA from your cheek cells available for the
molecular experiment by lysing the cells with a lysis solution.
We will then prepare the reactions and amplify the TAS2R38 gene on the
StepOne® or StepOnePlus® instrument. When amplifying your DNA, we
will use two different dyes: VIC® (for the C) and FAM™ (for the G) dyes.
The colors of these dyes cannot
by seen by the naked eye, but
the StepOne® or StepOnePlus®
Real-Time PCR Systems
will be able to read the color
information and group all of
your samples accordingly: blue
(GG), green (GC), and red (CC).
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Step-by-step genotyping
Each student will do his or her own DNA sampling. To perform these steps you will
need the following supplies:
— 1 tube (1.5 mL)
— 1 swab stick
— Lysis solution (150 µL)
— Stabilizing solution (150 µL)
— 1,000 µL pipettor with sterile tips
— Heating device (heating block for 1.5 mL tubes, or water bath)
— Permanent pen to write on plastic (Sharpie marker type)
Getting DNA samples:
1. Take a 1.5 mL tube and label it on the cap and on the side with your
initials.
2. Pipette 150 µL of lysis solution into the 1.5 mL tube that you just labeled.
3. Take a sterile swab by the wood or plastic end.
4. Put the cotton end on the inside of your cheek. Rotate and brush the
swab for about 20 strokes.
5. Immerse the swab in your 1.5 mL tube (the one with your initials on it).
6. Rotate the swab in the solution 5 times.
7. Lift the swab out of the lysis solution, and then press the swab against
the side of the tube to squeeze out the liquid.
8. Dispose of the swab in the trash and close your tube.
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Lysing the cells
Your cells contain DNA. In order to perform PCR, the DNA must first be released from the cells. This
is done by rupturing the cells, which is called lysis, allowing the DNA to spill out.
1. Make sure your tube is closed properly and labeled with your initials.
2. Set the heating device (heating block or water bath) to 95ºC.
3. When the heating device reaches 95ºC, insert your tube for 3 min.
4. Remove your tube from the heat. (Be careful. The tubes are very
hot!) Let it cool at room temperature for 1 min.
Stabilizing the DNA samples
Once the cells are broken and DNA is released, we need to
neutralize the solution. If lysis continues, the DNA will be degraded.
To stop the lysis reaction, we add the stabilizing solution. Here’s
how:
1. Recover your tube that is now at room temperature.
2. Thoroughly mix the DNA stabilizing solution.
3. Open your tube.
4. Add 150 µL of DNA stabilizing solution to your tube.
5. Pipette up and down slowly 15 times in your tube to mix the solution and your DNA sample.
6. Make sure your pipette is empty, and close the tube.
The tube can now be stored at 4ºC for a few days. If you are not going to proceed to the
rest of the experiment within a week, store the tubes at –20ºC.
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Genotyping experiment
This experiment should be done in teams of 4 or 5 students. Each team will be assigned a number. To
conduct this experiment you will need the following supplies:
— 1 tube (1.5 mL) (1 tube per team)
— TaqMan® GTXpress™ Master Mix (20 µL)
— TaqMan® SNP Genotyping Assay (40X) (188 µL)
— Nuclease-free water
— 1 96-well Fast plate per class
— 1 optical adhesive plastic film cover
— Pipettors with sterile tips to pipette volumes from 1.25 µL to 100 µL
— Centrifuge
— Applicator tool or thick plastic card (similar to a credit card)
Genotyping reaction setup
1. Thoroughly mix the TaqMan® GTXpress™ Master Mix bottle. Avoid creating bubbles.
2. If your DNA sample from the previous step is in the freezer, thaw it along with the
TaqMan® SNP Genotyping Assay.
3. Label a new 1.5 mL tube with your team number.
4. The following table indicates the quantities of the reagents that must be mixed together for
each genotyping reaction. You will need 1 reaction per student, plus 1 reaction for the negative
control,* and 1 extra reaction. You therefore need to multiply the numbers in the second
column by the number of people in your team plus 2. So for example, if there are 5 people in
your team, you will multiply by 7 (5 + 2 = 7) all the numbers in column 2.
*Negative control: A reaction that has all of the reagents, but no DNA. There should be no amplification in a negative control. If there is amplification, it means that you have contaminated your reaction and the other reactions might also have been contaminated, giving you incorrect data.
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5. Close your tube and vortex it for 3 seconds. If you don’t have a vortexer,
pipette the reaction mix up and down 10 times.
6. On the 96-well plate, in the column corresponding to the team number,
each student fills one well with 15 μL of the PCR reaction mix from step 4.
7. You should fill one well per student, plus one extra well for the negative
control. Make sure the reaction mix is in the bottom of the wells.
8. Each student will add 5 μL of their DNA to their well.
9. Record which student’s DNA is in each well on the following plate diagram
by writing your initials on the corresponding well. Each team should have a
negative control well (reaction mix with no DNA) at the end of its column or
row.
PCR reaction mix
Reagent Reagent volume/reaction
x _____ (number of people in your team + 2)
TaqMan® GTXpress Master Mix 10.0 μL
TaqMan® Genotyping SNP Assay (40X) 0.5 μL
Nuclease-free water 4.5 μL
Total 15.0 μL
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ExampleTeam 1 is Alex L., John P., Anne F., Shirley A., and Sam P.
A.L.
J.P.
A.F.
S.A.
S.P.
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10. Separate the white and transparent plastic parts
from an optical adhesive cover.
11. Place the transparent part over your plate and
make sure it covers the entire plate.
12. Seal the cover by pressing firmly on it with the
applicator tool (plastic rectangle). Do not write on
the adhesive cover.
13. Place your plate in a centrifuge and spin for 30
Correct! Incorrect
Liquid is at the bottom of the well
Not centrifuged with enough speed, or for enough time
seconds at 2,000 rpm. After the spin, all liquid should be at the bottom of the wells. Note: If you
do not have a centrifuge, you can use a salad spinner instead. Use zip ties to attach your plate
with the bottom of the wells facing out. Make sure to put an empty plate on the other side of the
spinner to balance it.
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1. Open the instrument.
2. Open the drawer by pulling it.
3. Load your plate onto the block.
4. Close the drawer by pushing it back.
Starting the StepOne® or StepOnePlus® Real-Time PCR Systems
5. On the computer, from the three options
choose Quick Start.
6. Enter the experiment name and select
“Genotyping” and “Fast”.
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9. Now we are waiting for the instrument to amplify DNA and compute the results.
When the run is completed, the instrument and computer will save the results
automatically. A short period of time after the completion of the run, both the
instrument and computer will go into sleep mode to save energy.
7. Click on the green “Start” button in the
bottom right corner.
8. The next screen looks something like this.
10. Open your experiment file. We are going
to tell the software where the samples
are. From the Experiment Menu, click
on “Setup” and then “Plate Setup” in the
sub-menu.
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11. To add sample names, click on “Add Sample”
and add as many samples as there are
students in the class. Rename each sample
number with the student’s name.
12. Using the plate template on page 11, assign
names to wells on the plate by highlighting
a well on the plate layout (right side of
screen), then selecting SNP assay 1 and the
corresponding student’s name.
Results analysis
Once the wells have all been named, click on
the “Analysis” menu and select the “Allelic
Discrimination” sub-menu. Next click on the
green “Analyze” button in the top right corner of
the screen.
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You should now see 3 clusters of colored dots on
the left half of the screen. To view the group to
which you belong, zoom in on the plate layout.
Blue represents GG, or the high tasters; green is
GC, or the average tasters; red is CC, or the non-
tasters; and the black X represents the negative
control.
Take another look at the spreadsheet you filled out when you tasted the PTC strip. Compare the
results from the strip tasting and the molecular reaction. How well do they match? What are your
thoughts on why some results don’t match up?
Once you have
your results,
discard all DNA
samples.
Notes for teachers:• PTC strip tasting results can be inaccurate if your mouth is dry or you just ate or drank
something.
• The distinction between tasting a lot and a little can be subjective.
• You could have contaminated your DNA sample with your neighbor’s.
• There are 3 SNPs in the PTC-tasting gene. The other two may not be driving your taste perception in the same direction.
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