SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs):

Small markers of significant information

Lori Renehan
BTech 1010-001

The question of whether or not a person could taste the bitterness of phenylthiocarbamide (PTC)
because of a SNP in their gene TAS2R38 was put to the test. Cheek cells were extracted and
suspended in water. Using PCR, a short section of the TAS2R38 gene was amplified, and then
digested with enzyme HaeIII. The resulting fragments were separated using 2% agarose gel and
electrophoresis. The SNPs were identified by the numbers of base pairs (bp) and a prediction was
made as to whether or not the person would be able to taste PTC. An actual taste test was
performed following the analysis, and all but one student found that the results of the analysis
did correspond to their ability to taste PTC. SNPs are viewed as the central cause of human
genetic variability and are proving indispensible in personalized medicine.

Introduction
The human tongue contains between 2000
and 5000 taste buds, with approximately 50-
100 taste cells per taste bud. Taste buds are
used to identify hundreds of flavors that are
categorized into five groups: bitter, salty,
sour, sweet, and umami. A sweet flavor
indicates a source of energy, and salty a
source of the vital mineral, sodium chloride.
Umami, coming from a Japanese word for
deliciousness, detects glutamate, found in
Parmesan cheese, wild mushrooms, and soy
sauce, typically guiding us toward a source
of protein. Tasting something sour indicates
the presence of acid, which can be beneficial
in the form of fruit or yogurt. It can also
indicate unhealthy, rotting food. A bitter
taste is considered unpleasant and can warn
us of a variety of harmful substances.
Unfortunately, many useful medications are
also bitter tasting and require special
packaging to deliver them past discerning
taste buds.
1


Typically, a human has around 30 types of
bitter taste receptors. Taste receptor, type 2,



1
http://ghr.nlm.nih.gov/handbook/genomicresearch
/snp; http://www.everydaylife.globalpost.com
member 38 (TAS2R38) controls the ability
to taste the bitter-tasting compound found in
plants of the Brassica sp, as well as the
synthetic compounds phenylthiocarbamide
(PTC) and 6-n-propylthiouracil (PROP).
One single nucleotide polymorphism (SNP)
will determine if a person is able to taste
PTC or not.
2


When a cell divides it makes a copy of its
DNA. Mutations occur when the copy is not
perfect. There are three common types of
mutations:

Substitution Insertion Deletion
.

Ajay Saini, a plant molecular biologist,
informs us that if the frequency of a
variation in at a particular locus in a
population is less than 1% it is considered a
mutation . . . and . . . if it occurs more than
1% it is termed as a SNP.
3


A SNP in the gene TAS2R38 is found on the
C allele. It could read GGCGGGCACT
or GGCGGCCACT. The first order of base

2
http://www.ncbi.nlm.nih.gov/gene/5726
3
www.researchgate.net
pairs indicates the recessive trait with the
inability to taste PTC, the second order
indicates the dominant trait for tasting PTC.

In this experiment a polymerase chain
reaction (PCR) was used to amplify the
exact section of the gene containing the
SNP. Kary Mullins developed this method
in the 1980s. DNA polymerase, with the
aid of a primer, is used to duplicate a
specific template of DNA millions of times
over.
4


After obtaining the PCR products, HaeIII
was used to digest the sample. The results
were analyzed by gel electrophoresis to
determine the base pair (bp) sizes. The gel
was then viewed using transillumination and
a photograph was taken.

Materials and Methods

A 1.5 mL centrifuge tube and a paper cup
were labeled. A mouthful of water swished
around for 30 seconds, was used to obtain
cheek cells. The water and cell solution was
expelled into the paper cup. The cup was
swirled gently to distribute the cells evenly.

1000uL of the solution was transferred to
the 1.5 mL tube and placed in a balanced
configuration with other students tubes in a
microcentrifuge and spun for 90 seconds at
full speed. The supernatant was poured off,
without disturbing the cell pellet at the
bottom of the tube. The cells were
resuspended with 30 uL of the solution from
the cup. After popping any bubbles, 30 uL
of cell suspension was withdrawn and added
to a 1.5 mL centrifuge tube containing 100
uL of Chelex, capped and labeled.

4
http://www.ncbi.nlm.nih.gov/genome/probe/doc/
TechPCR.shtml

The sample was placed into a boiling water
bath for 10 minutes. Afterwards the tube
was flicked with the finger for 5 seconds.
The tube was placed again in the
microcentrifuge with other students tubes
and spun for 90 seconds at full speed. 30 uL
of supernatant was pipetted into a clean 1.5
mL tube. Debris and Chelex beads were to
be avoided. Cap and side were labeled and
the tube was stored on ice until
amplification could be performed.

For amplification of DNA by PCR method,
a PCR tube with Ready-to-Go PCR bead
was obtained and labeled. 22.5 uL of PTC
primer/loading dye was pipetted into the
tube. Time was allowed for the bead to
dissolve. 2.25 uL of cheek cell DNA was
pipetted into dye mix.

The tube was placed with other students
tubes into a thermal cycler and was
programmed for 30 cycles of the following
program:
Denaturing 94 C 30 seconds
Annealing 64 C 45 seconds
Extending 72 C 45 seconds

A 1.5 mL tube was labeled and marked with
a U for undigested, and 10 uL of PCR
was pipetted into the tube. This tube was
stored on ice until further use. 1 uL of
HaeIII was pipetted into the remaining PCR
product and labeled D for digested.
Reagents were mixed and pooled by pulsing
in a microcentrifuge or by pipetting them up
and down. The tube labeled D was placed
with other students samples in a thermal
cycler and was digested at 37 C for 30
minutes.

During digestion a 2% agarose gel was
prepared with a 7-well comb and allowed to
set for 20 minutes. The comb was removed;
the gel was set into the chamber and covered
with 1x TBE buffer.
The marker and samples were pipetted in the
wells in this order:

MARKER STUDENT 1 STUDENT 2
pBR322/ U D U D
BstNI



The gel was run at 130 V for about 30
minutes, then was removed and
photographed.

Results

The results of the experiment revealed that
both Student 1 and 2 had fragments at 221
bp, 177 bp and 44 bp, which indicated that
we were heterozygous (Tt) for tasting PTC.
See Fig. 1.

After the DNA analysis, the students took a
taste test using a control strip followed by a
PTC paper to verify the results of the
analysis. The taste test for Students 1 and 2
did verify the results of the DNA analysis,
that they were heterozygous PTC tasters.

Lanes 1 2 3 4 5 6


FIG. 1 Photograph of electrophoresis gel. Lane one
is the marker , with at least four visible bands,
followed by the undigested and digested DNA of
Students 1 and 2, in lanes 2,3, 5 and 6 respectively.
The digested DNA has two or three distinct bands
proceeding down from the wells.


Discussion

There were three possible results to be
concluded from the DNA analysis. A
student could have two dominant alleles
(TT) that were homozygous for tasting PTC,
one dominant and one recessive allele (Tt)
heterozygous for tasting, or two recessive
alleles (tt) homozygous for non-tasting.
DNA for homozygous dominant would
show in two bands of 177 bp and 44 bp.
DNA for homozygous recessive would show
in a single band at 221 bp.

It is believed that the ability to taste
bitterness evolved as a way for early humans
to detect poisonous plants. Although PTC is
not found in nature, a PTC taster will have
the ability to taste similarly bitter
compounds found in nature that often are
toxic.
5


Studies are suggesting that a PTC non-taster
may have a polymorphism to taste a
currently undiscovered bitter compound. It
is therefore more beneficial to have a
mostly heterozygous population with
flexibility in their bitter taste perception,
enabling them to avoid a greater number of
toxins than either homozygotic group.
6


This experiment is one example of how
informative a SNP can be. SNPs can affect
how humans develop diseases and respond
to treatments. SNPs help scientists locate
genes that are connected with a certain
disease. Noted in an article in Modern
Pathology, a SNP array was used to identify
chromosomal changes connected with
tumors on the kidneys.



5
http://learn.genetics.utah.edu/content/inheritance
/ptc/
6
wikipedia.org/wiki/TAS2R38
Of twenty tumors, nineteen were identified
using this method.
7
SNPs are also used for
personalized medicine in humans and
breeding programs with crops and
livestock.
8


7
http://www.nature.com/modpathol/journal/v21/n5
/abs/modpathol200820a.html


8
wikipedia.org/wiki/Single-
nucleotide_polymorphism;http://ghr.nlm.nih.gov/ha
ndbook/genomicresearch/snp