-Elina Shrestha
Tasting Phenylthiocarbamide (PTC) –Restriction Fragment Length Polymorphism (RFLP)
Analysis of student Phenotypes and Genotypes at the PTC Gene.
There exist many individual differences in terms of how one perceives his environment,
for example, in sensing various chemicals. Studies in the past have shown that polymorphisms in
human sensory receptor genes could alter perception by coding for functionally distinct receptor
types (Bufe et al., 2005). In relation to this, phenylthiocarbamide (PTC) or phenylthiourea is an
organic compound known to have the unusual property of tasting very bitter to some people
whereas being virtually tasteless to the other. The ability to taste PTC primarily depends on the
genetic makeup of the taster. It is controlled by the TASR238 taste receptor gene, also known as
PTC gene, which is located on the long arm of chromosome 7 (7q34) and is about 1003 base
pairs in length. It has been reported that sequence variants (polymorphism) in this gene correlate
with differences in bitterness recognition of PTC. The three most common occurrences of
polymorphisms in TAS2R38 are at amino acid position 49, where either a proline or an alanine is
encoded, at position 262, where either an alanine or a valine is encoded, and at position 296,
where either a valine or an isoleucine is encoded. The two commonly found haplotypes are PAV
and AVI, whereas the haplotypes AAI, PVI, and AAV do not occur frequently. Therefore, there
are two alleles that are mostly found coding for amino acid with haplotypes PAV and AVI (Bufe
et al., 2005). The variation in the ability to taste PTC appears to be widely studied as a
Mendelian trait. In the past, many surveys regarding the genetics of taste thresholds were
performed to classify the tasters and non tasters of PTC roughly according to their taste acuity by
means of dilutions at which the bitter taste was first detected. By analyzing the statistical report
on the inheritance of the trait from the parents to their children, Blakeslee found that the inability
to taste PTC was inherited as a Mendelian recessive (Blakeslee, 1931). Therefore, the taster
allele (T) is always dominant and the non-taster allele (t) always recessive. A person’s ability to
taste PTC varies with the type of alleles she receives from her parents. There are three possible
combinations of the two alleles one can get from her parents. A person either receives two
dominant taster alleles(TT), one from each parent making her a homozygous dominant; or two
recessive non-taster alleles (tt) making her a homozygous recessive; or a dominant allele from
one parent and a recessive allele from the other (Tt) that makes her a heterozygous. Though the
first and the third cases are genotypically different, since both have at least one copy of the
dominant allele, they are phenotypically alike i.e. both are tasters of PTC.
The phenotype of a person only tells us whether she is a taster or a non-taster and is,
therefore, easily determined. In contrast, the genotype analysis requires us to understand the
genetic make up of an individual at the molecular level and the type of alleles involved which is
more demanding. The goal of our project is to identify both phenotype and genotype for the PTC
gene and compare the results from both analyses. The two alleles differ at three nucleotide
positions 145, 785 and 886 in the PTC gene. With the use of a suitable restriction enzyme that
can cut only the dominant allele at a specific site, it is possible to identify one’s genotype for the
PTC taste receptor gene. The single nucleotide polymorphism at position 785 plays an important
role in recognizing the genotype. For a dominant allele, the restriction enzyme Fnu4H1
recognizes the restriction recognition site at C785 and cuts there, whereas this site is completely
absent in the recessive allele where C is replaced by T. Thus, only the dominant allele gets
digested and broken apart into two fragments. By choosing the appropriate primers that flank the
polymorphic Fnu4H1 site, Polymerase Chain Reaction can amplify only 303 bp long fragments
of the PTC gene receptor from position 547 to position 849. When digested with Fnu4H1
restriction enzyme, the dominant taster allele gives two fragments, 239 and 65 bp long. On the
other hand, the recessive non-taster allele remains undigested due to the lack of restriction
recognition site for the enzyme. As such, the amplified fragment of 303bp length for this allele
will not be divided any further. The three different fragments can be easily separated by gel
electrophoresis and the specific allelic combination or the genotype for the PTC gene of any
individual can be identified by observing the positions of bands. A non-taster homozygote will
only produce fragments 303 bp long since it has two recessive alleles which do not get cut by the
restriction enzymes. Hence, it will have only one bright band of 303 bp long fragments. A taster
homozygote, in contrast, will give two fragments of lengths 65 bp and 239 bp as both of his
dominant taster alleles get digested by the added enzyme. However, if an individual is a
heterozygote, her digested PCR product will generate all three bands, 303 bp band from the
recessive allele fragment, and bands at 65 bp and 239 bp from the digested taster allele
fragments. If a person has the ability to taste phenylthiocarbamide, she could either be a
heterozygous or a homozygous dominant whereas a person who is a non taster is always a
homozygous recessive. In our lab, we are trying to confirm this relationship between phenotype
and genotype for the PTC gene of any individual by experimenting on our own individual DNA.
Methods and Materials:
Determination of PTC-tasting phenotype and Isolation of Genomic DNA: The taste of
small strips of PTC control paper and the PTC taste paper was recorded and everyone in the lab
was grouped into tasters and non-tasters. In order to isolate the genomic DNA, some cheek cells
were suspended in 200 µl of 5% Chelex buffer contained in a 1.5 ml microfuge tube and 2 µl of
10mg/ml Proteinase K was added into it followed by 25minutes incubation at 56 oC and 8minutes
boiling to 100 oC. The resulting solution was centrifuged and the concentration of the supernatant
containing our extracted DNA was measured using a Nanodrop spectrophotometer.
Polymerase Chain Reaction Set up: In order to amplify the PTC gene sequence of the
extracted DNA, a master mix of ddH2O, 10mM dNTPSs, 25mM MgCl2, 10X PCR Buffer,
forward and reverse primer and Taq DNA polymerase was made in 1.5 ml microfuge tube.
Aliquots of 43.5 µl from the master mix were dispensed into small PCR tubes and each student
added 6.5 µl of the supernatant from her DNA into the individual PCR mix. Then the PCR tubes
were placed in the thermal cycler set for one cycle of 10 minutes in 950C and 5 minutes in 550C,
40 cycles in 72 0C, 45 seconds in 950C and 45 seconds in 550C , and one cycle of 10 minutes in
720C and in 40C until collected. The reaction tubes were then stored at -200C after the
completion of all the cycles.
Set up of restriction digest of Amplified PCR product: Another master mix of 24 µl of
ddH2O, 12 µl of NEB RE buffer #4(10X) and 4 µl of Fnu4H1 Restriction Endonuclease was
made and to each 10 µl aliquot was added individual 20 µl PCR product that contained the PTC
gene sequence of an individual student. The mixture was then subjected to incubation overnight
in a 370C waterbath.
Separation of the restriction enzyme digest and Purification of undigested DNA: In a 1.5
µl microfuge tube containing 3 µl 5X loading dye, restriction digest was added and mixed. The
25 ul undigested PCR product is then mixed with 1ul of loading dye. We then loaded both dyed
restriction digest and undigested PCR products, and also the DNA ladder into the designated
wells on the low melting electrophoretic gel. After setting these up, the gel was electrophoresed
at 100volts for ~30 minutes and was imaged using ultraviolet source. The DNA band in the gel
was cut out for each student and the gel slice was treated with QG buffer and heated in a 650C
heat block for 10 minutes. This was followed by the addition of isopropanol; the mixture was
transferred to a spin column placed in a collection tube and centrifuged for a minute. The spin
column was first washed with QG buffer and then with the PE buffer and finally dried.was sent
out for sequencing. The DNA was then collected in a microfuge tube by eluting with 30ul of TE
buffer and its concentration was determined by using the Nanodrop spectrophotometer. The
DNA samples were then shipped to the biotechnology company for sequencing.
Analysis of sequence data using bioinformatics (BLAST searches): The isolated DNA
sequences of our PTC gene were compared to sequences in a bioinformatics database. This was
done by reconstructing the gene through the computer program LaserGene and then performing a
BLAST search of the gene. We compared a given person’s available PTC gene sequence with
the most similar gene sequences of both human taster and non-taster on the Blast search.
Calculation of allele frequencies for the PTC gene based on the Hardy-Weinberg
equilibrium: Given the lab’s phenotypic and genotypic data, we used the Hardy-Weinberg
equilibrium equation ( p2 + 2pq +q2 = 1, where p is the frequency of the dominant allele and q
is the frequency of the recessive allele for a given trait) to determine the probable genotype
frequencies for tasters and non tasters within the lab group. Similar calculations were done for
the whole class population. The calculated determination was also compared with the RFLP
determination.
Results:
Determination of PTC tasting phenotype: The PTC control paper was tasteless to the entire lab
group whereas the taste of the PTC taste paper was bitter for most of the people in the lab with
only two lab members as non-tasters. Three of us, Sweta, Shala and I worked together and Sweta
and I were classified as tasters, and Shala as a non-taster according to our ability to taste the PTC
taste paper.
Determination of PTC tasting genotype : The agarose gel loaded with Shala’s, Sweta’s and my
digestested and undigested PCR products, that was electrophoresed was imaged using ultra violet
light as shown in figure 1. Distinct bands at 303 base pair position were observed in lanes 2, 4
and 5, those corresponding to the undigested PTC genes of me, Shala and Sweta respectively.
Shala’s digested product showed only one faint band at 303 base pair whereas no distinct bands
were seen for Sweta’s undigested products and mine as well. From this, we inferred that Shala
was a homozygous recessive, and therefore a non-taster. Since the gel electrophoresis results
were unclear for me and Sweta, it was hard to infer whether we were homozygous dominant or
heterozygous.
.
Figure 1- Electrophoretic separation of undigested and digested PTC receptor genes from me,
Shala and Sweta from the left. Lane 1 contains the DNA ladder; lanes 2, 4, 6 contain undigested
PCR fragments and lanes 3,5,7 contain digested PCR fragments for me, Shala and Sweta
respectively.
Calculation of allele frequencies for the PTC gene based on the Hardy-Weinberg equilibrium:
For our lab section, phenotypic data showed that there were 7 tasters and 3 non-tasters.
By using this data together with the Hardy-Weinberg equilibrium equation, the phenotypic
frequencies for tasting PTC and allele frequencies for the PTC gene were determined within the
lab group. The frequencies of the dominant allele and the recessive allele were found to be equal
to 0.45 and 0.55 whereas Hardy-Weinberg equilibrium equation suggested that the population of
10 people should have 7 tasters and 3 non-tasters when it reaches the equilibrium in the course of
evolution. Similarly, the genotypic data contained one homozygous dominant, 3 heterozygous
and 3 homozygous recessive which produced the exact same numbers at equilibrium. Also, the
frequencies of dominant and recessive alleles were found to be 0.36 and 0.64 respectively.
When the whole class population was taken into account, 84 had taster phenotype and the
rest 28 had non-taster phenotype. The frequencies of both dominant and recessive alleles were
equal to 0.5 and the calculated equilibrium population constituted 28 homozygous dominant, 56
heterozygous and 28 homozygous recessive. The genotypic data indicated the presence of 19
homozygous dominant, 38 heterozygous and 15 homozygous recessive. From this data, the
frequencies of dominant and recessive alleles were determined to be 0.53 and 0.47 respectively
and 20 homozygous dominant, 36 heterozygous and 16 homozygous recessive were found to
coexist at equilibruim.
Analysis of sequence data using bioinformatics (BLAST search): Since we did not get a
good amplification of our DNA sequence from PCR reaction, we used the PTC gene sequence of
one of the individuals from our class as our Query in BLAST search and compared it with the
sequences displayed in the bioinformatics database. The summary of the BLAST search results
are shown here below:
Individual’s name:
ALL
Genotype:
TT (taster phenotype)
Comparison with PTC Taster Allele
E-Value
=
3.7 * 10 ^ -156
Score
=
558
% Query =
100
Mutations:
‘-‘ in place of A at 247 bp
‘-‘ in place of A at 250 bp
R in place of A at 253 bp
Comparison with PTC Non-Taster Allele
E-Value =
8.6 *10 ^ -145
Score
=
521
% Query =
98.6
Mutations:
G in place of A at 57 bp
Discussion:
The ability to taste the bitter chemical phenylthiocarbamide has been found to be
absent in some people as indicated by our class phenotypic data. Such variation in human
population is a consequence of polymorphism in TAS2R38 (PTC) taste receptor gene. In our lab
we studied the relationship between our phenotype, as tasters and non tasters, and genotype as
either homozygote(dominant or recessive) or heterozygote. Since the PTC taster allele is a
dominant allele and I could taste the bitterness, we predicted two genotypes, either a
homozygous dominant taster (TT) or a heterozygote taster (Tt). The presence of atleast one
dominant allele would enable me to taste PTC. In order to determine my genotype, my DNA
extract was digested by recognition enzyme and were analyzed by electrophoretic separation of
digested and undigested PTC receptor genes. Since the electrophoresed gel did not produce a
clear image so as to be able to distinguish the bands of digested products, it was hard to conclude
anything about my genotype. This might have occurred due to some pipetting errors since we
were dealing with very small quantities of reagents in microliter units. Also, the faint bands are
indicative of the lack of proper amplification due to inadequate reagents used to carry out the
reaction. However, the same gel showed some faint yet distinguishable bands from Shala’s
digested and undigested products, both at 303 bp indicating that she was a homozygous recessive
and thus a non-taster. Since she was unable to taste the bitterness of PTC, it was predicted that
the only possible genotype was a homozygous recessive for PTC gene. The presence of only one
band at 303 bp for the digested product implies that the restriction enzyme Fnu4H1 has failed to
cut the 303 bp PTC gene fragment due to the absence of restriction recognition site at nucleotide
position 785 (where C is replaced by T) on either alleles (tt). The phenotypic and genotypic
results are therefore in accord with each other. All the other three lanes associated with the
undigested products produced bands at 303 bp position suggesting that, prior to the addition of
restriction digest, the PCR fragments were 303 bp long and were not cut. Therefore, they served
as controls.
Of the entire sequenced PTC genes, BLAST search was performed on the data of an
individual named ‘All’. Electrophorectic gel analysis had shown that she was a homozygous
dominant with TT taster alleles. On comparing her sequence a taster allele and a non-taster allele
from two hit BLAST searches, a 100 % query result with a taster allele and 98.6% query result
with a non taster were seen. The E-value for the taster allele was much higher than for the nontaster; All’s alleles were much more similar with the taster allele than the non-taster allele.
Phenylthiocarbamide is not a natural compound; it is an organic compound containing
isothiocyanate. In nature there are edible (vegetables such as cabbage, broccoli and Brussels
sprouts) and non-edible substances containing this compound. Most of the bitter tasting things
are harmful for health; PTC receptor could be a defense mechanism of the body against such
toxins. (Wooding et al, 2004) A person’s ability to taste PTC might protect her from toxic
products related to such taste. (Floriano et al., 2006) The ability to taste PTC and liking for
certain types of food influences the diet of a person which in turn affects health. (Floriano et al.,
2006)Adequate knowledge of PTC and other taste receptors help in better understanding of the
relationship between human diet and health.
Literature Cited:
1
2
Bufe, B., Breslin, P.S., Kuhn, C., Reed, D.R., Tharp, C.D., Slack, J.P., Kim, U., Drayna,
D., Meyerhof, W., 2005. The Molecular Basis of Individual Differences
in Phenylthiocarbamide and Propylthiouracil Bitterness Perception. Current Biology, 15:
332-337.
Blakeslee, A.F., 1932. Genetics of Sensory Thresholds: Taste for Phenylthiocarbamide.
Proc. N. A. S. 18: 120-130