The document summarizes a lab experiment comparing participants' genotypes and phenotypes for tasting the bitter compound phenylthiocarbamide (PTC). The results showed that the genotype determined by PCR and gel electrophoresis matched the self-reported PTC tasting phenotype for all participants. Specifically, those with the tt genotype could not taste PTC, while those with the TT or Tt genotypes reported PTC as bitter tasting. The lab demonstrated that an individual's ability to taste PTC is inherited in a dominant/recessive manner governed by the TAS2R38 gene.

1. PTC Lab 1
Phenylthiocarbamide Tasting: Comparing Genotype and Phenotype
Cammie Coffey

2. PTC Lab 2
INTRODUCTION
Tasting is a two step process between taste cells and sensory neurons in the brain. First, a
taste molecule binds to a bitter taste receptor. Then, stimulation of this receptor sends a signal to
the brain. There is also an inherited component to how people taste. Understanding how genetics
is related to the bitter taste of phenylthiocarbamide (PTC) is important. It can determine how
knowing a person's genotype can be used to predict their reactions to certain drugs.
In the 1930s, a chemist named Arthur Fox synthesized some PTC. When some of the
dust became airborne, only certain people noticed a bitter taste in their mouths. This initiated the
study of taste being genetically linked. Albert Blakeslee conducted successive testing at
Carnegie Department of Genetics. These tests revealed that a recessive (tt) trait in people cause
them to be unable to taste PTC, the ability to taste PTC is dominant (TT), and heterozygous (Tt)
individuals can somewhat taste PTC. It was not until 2003, that the PTC taste receptor genes,
TAS2R38, was identified.
In the human population, there are three nucleotide positions, and each variable position
is called a single nucleotide polymorphism (SNP). A better understanding of cell surface
molecules, and their influence over the activity in pharmaceuticals has many potential health
applications. One example is how SNPs in serotonin transporter and receptor genes can predict
undesirable responses to anti-depressants.
The purpose of the PTC lab is to compare a person's bitter tasting phenotype to their
genotype, to demonstrate the process of amplifying and analyzing DNA through polymerase
chain reaction (PCR), and to understand SNPs and their use in predicting drug response.

3. PTC Lab 3
The hypothesis is that the each student's genotype via gel electrophoresis will match their
phenotype determined by the tasting strips. The null hypothesis is that the genotype and
phenotype results will not coincide.
MATERIALS AND METHODS
 0.9% saline solution, 10 mL
 10% Chelex®, 100 𝜇L
 Permanent marker
 Paper cup
 Micropipets and tips (10-1000 𝜇L)
 Micropipets and tips (1-100 𝜇L)
 2 Microcentrifuge tubes 1.5-mL
 Microcentrifuge tube rack
 Microcentrifuge adapters
 Microcentrifuge
 Thermal cycler
 Container with crushed ice
 PTC primer/loading dye mix, 22.5 𝜇L
 Ready-To-Go™ PCR beads (in 0.2-mL or 0.5-mL PCR tube)
 Restriction enzyme HaeIII 10 𝜇L
 pBR322/BstNl marker
 1x TBE, 300 mL
 0.5 g of agarose

4. PTC Lab 4
 50 mL of 1x TBE
 5µL of 1x GelGreen
 Gel electrophoresis chamber
 Power supply
 Staining trays
 Latex gloves
 UV transilluminator
 Scotch tape
 PTC taste paper
 Control taste paper
Start isolating the DNA by labeling a 1.5-mL microcentrifuge tube and paper cup with an
assigned number. Put saline solution into mouth and rinse cheek pockets for 30 sec. Spit the
solution into the paper cup. Swirl the solution to mix the cells, and transfer 1000 µL of the
solution into the labeled tube. Place the tube into the microcentrifuge and spin at full speed
for 90 sec. Pour the supernatant off into the paper cup and do not disturb the cell pellet at the
bottom of the tube. Using a micropipet set to 30 µL, resuspend the cells in the remaining
saline. Remove 30 µL of the cell suspension, add it to a PCR tube with 100 µL of Chelex and
label it with the assigned number. Put the PCR tube into the thermal cycler set for one profile
99º for 10 min. When the profile is complete, vigorously shake the PCR tube for 5 sec. Put
the tube into the microcentrifuge and spin at full speed for 90 sec. Use a fresh tipped
micropipet and transfer 30 µL of supernatant into a new 1.5-mL tube, label the cap and side
of the tube with the assigned number and store on ice until the next phase.

5. PTC Lab 5
Start amplifying DNA by getting a PCR tube with a Ready-To-Go™ PCR Bead and label
it with the assigned number. Use a fresh tipped micropipet to add 22.5 µL of PTC
primer/loading dye into the tube and allow the bead to dissolve. Use a fresh tipped
micropipet and add 2.5 µL of the cheek cell DNA into the primer/loading mix, making sure
no cheek DNA is left in the tip. Store on ice until the thermal cycling phase. Put the PCR
tube into the thermal cycler programmed for 30 cycles, denaturing at 94º for 30 sec,
annealing at 64º for 45 sec, and extending at 72º for 45 sec. Remove the tube and store it on
ice until the next phase.
Start digesting the PCR product with HaeIII by labeling a 1.5-mL tube with the assigned
number and a "U" (undigested). With a fresh tipped micropipet, put 10 µL of the PCR
product into the "U" tube and store it on ice until the next phase. Using a fresh tipped
micropipet to place 1 µL of HaeIII into the PCR tube with the remaining PCR product and
label it with a "D" (digested). Mix and pool reagents by pulsing it in a microcentrifuge. Put
the PCR tube into the thermal cycler set to one cycle of digesting at 37º for 30 min. Remove
the tube and store it on ice until the next phase.
To analyze the PCR products by gel electrophoresis, start by sealing the gel-casting ends
and adding a well-forming comb. Cover about half the height of the open teeth of the comb
with 1% agarose solution and allow the gel to solidify completely. Add 1x TBE buffer to
cover the surface of the gel and place into the electrophoresis chamber. Remove the comb
and fill in the wells with more 1x TBE buffer. In the far left lane of the gel, use a micropipet
to place 20 µL of pBR322/BstNl size markers. With a fresh tipped micropipet, place 10 µL
of "U" and 16 µL of "D" into separate wells and run the gel at 90V for 30-40 min. Remove
the gel from the electrophoresis chamber and view the results using transillumination. To

6. PTC Lab 6
understand the results, a tt non-taster will show a single band in the same position as the
control. A TT taster will show two bands of 177 bp and 44 bp ahead of the control. A Tt
taster will show three bands of 221 bp, 177 bp and 44 bp. It is common to see a fuzzy band
ahead of the 44 bp. This is the "primer dimer" and it verifies all components were on hand for
amplification.
To determine the phenotype taste 2 taste papers, one with PTC (sample A) and a control
without PTC (sample B). If the taste paper with PTC taste bitter, the phenotype prediction is
a taster TT, a mildly bitter taste is Tt. If no difference is detected between sample A and
sample B the phenotype prediction is tt.
In this lab there are 13 participants. Collect the phenotype and genotype data of all the
participants to determine how accurately the TAS2R38 genotype can predict PTC-tasting
phenotype.
RESULTS
After placing the taste papers on the tongue, sample A tasted very bitter, while sample B
tasted like paper. The gel results included two bands on the digested sample and one band on the
undigested sample. The phenotype prediction is strong taster and the genotype is Tt. The results
for the groups phenotype prediction was 38% strong taster, 0% weak taster, and 62% non-taster.
The groups genotype results were 23% TT, 15% Tt, and 62% tt.
DISCUSSION
Determining the TAS2R38 genotype is accurate in predicting whether or not a person is a
PTC taster or non-taster. All the participants with the tt genotype reported tasting no difference

7. PTC Lab 7
between sample A and sample B. Everyone who tasted bitter on sample A had the genotype for
either a weak or strong taster. As the results show, the only disadvantage is in determining the
phenotype difference between a weak or strong taster. There is no way to determine the level of
bitter each participant experiences. What a person eats or drinks prior to the test can also affect
their perception of bitter. What this shows about classical dominant/recessive inheritance is that
to be dominant you only need one copy of the gene to taste bitter. To be a non-taster you need
two copies of the gene to be expressed. It can be overridden by a dominant form of the trait.
HaeIII discriminates between the C-G polymorphism by separating the GGCC sequence
in the TAS2R38 gene, while the non-tasters have a GGGC sequence that will not be separated.
On the taster allele an HaeIII site is created and no site is made on the non-tasters. The C-G
ploymorphism mutation in the TAS2R38 gene is a non-synonymous, missense mutation. The
amino acid is changed in the protein and is altered from proline (taster) to alanine (non-taster). It
is believed that since there are more non-tasters versus tasters, balancing selection could be
occurring. This would mean that a heterozygote might develop the ability to taste an unknown or
exotic bitter molecule, which could help them avoid potentially toxic compounds. Since a
heterozygote taste bitter weaker, this could be advantageous when it comes to eating fruits and
vegetables. Strong bitter tasters may avoid foods like cabbage or broccoli, where weak and non-
tasters may enjoy them. We fail to reject the null hypothesis because these phenotype predictions
coincide with the genotype results.