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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