BTEC 1010, 12/08/13
Michael Turley
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Utilization of a Single Nucleotide Polymorphism in Gene TAS2R38 as a Prediction of
Phenylthiocarbamide (PTC) Tasting Ability
Abstract
The ability to taste bitterness is a result of a specific genotype and was tested using multiple procedures analyzing DNA. The ability to taste PTC can be tracked back to a single change in DNA sequence of PTC tasting gene TAS2R38. This can be demonstrated by a simple experiment. Cells are collected with a saline mouth wash and the DNA is released by boiling.
The DNA can then be separated from the cell debris and other contaminants. Using PCR a small section of the DNA sequence from within the gene is isolated and amplified. HaeIII restriction enzyme is used to cut strands, if present, containing GGCC sequence into two fragments.
Strands with G-C SNP are not cut by the enzyme. Gel electrophoresis separates the fragments and allows analysis of differing fragment size measured in base pairs (bp). The use of strips of saturated phenylthiocarbamide (PTC) paper indicated the phenotype of an individual as a taster or non-taster. The genotype of a taster or non-taster was determined after the phenotype had been concluded. If PTC is tasted on the strip, the possible genotypes would be homozygous dominant
(TT) or heterozygous (Tt). If PTC is not tasted, the only possible genotype is homozygous recessive (tt).
Introduction
Single nucleotide polymorphisms (SNPs) are the most frequent occurring manifestation of genetic variation in the human genome. Approximately one out of every 1000 nucleotides in the human genome is expected to be a SNP site, accounting for more than 90% of all differences between humans. SNP’s contribute to the variation in human phenotypes, such as disease susceptibility, responses to drugs and environmental chemicals, and (Landegren et.al., 1998;
Twyman, 2004; Zhou et al. 2005). Identifying SNPs and finding functional changes in them is an important tool used today in molecular diagnostics and biology. At present, Real-Time PCR is the most used technology for detection of SNPs (Kwok, 2002).
Around 1930, in Dupont laboratory, Arthur Fox was doing some tests on phenylthiocarbamide (PTC) and some dust from the PTC became airborne. His lab partner, C.
R. Noller, complained that he tasted something extremely bitter. Fox could not taste the bitter from the airborne PTC and he then tasted the actual crystal of PTC and still could not taste bitter.
Single nucleotide polymorphisms (SNPs) are the most frequent occurring manifestation of genetic variation in the human genome. There are two alleles of the PTC gene, meaning individuals can carry two copies of the PTC taster allele, two of the non-taster allele, or one of each allele. Bitter taste is directed by TAS2R38 gene family members, which belong to the large family of seven Trans-membrane G-protein receptors (Drayna 2005). Like all phenotypic traits, sensitivity to PTC has a hereditary nature (Suzuki et. al 2010).

BTEC 1010, 12/08/13
Michael Turley
If an individual is able to taste PTC, it means he or she has a dominant allele and inherited it from either both of his or her parents, or from one parent (Wooding et. al 2004). If an individual is not able to taste PTC, he or she would be a homozygous recessive taster for PTC
(Wooding et. al 2004). These genotypes can be determined through amplifying, digesting and analyzing the DNA with Electrophoresis. The result will be indications shown by the size of the
DNA in base pair measurement.
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Materials and Methods
DNA cells were extracted by pouring 10 mL of 0.9% saline solution into the analyst mouth and vigorously swishing and massaging outer cheeks for 30 seconds. The saliva and saline mixture was then expelled into a plastic cup. 1000 µl of the saliva and saline mixture was then transferred into a 1.5 mL micro centrifuge tube. The tube was then spun in a centrifuge for
90 seconds at 13,000 rpm. The supernatant was then discarded and the pellet was resuspended in remaining supernatant by pipetting in and out with a micropipette. The analyst withdrew 30 µL of cell suspension and added it to a 1.5 mL tube containing 100 µl of chelex. The tube was then placed on a thermal cycler set at 99° F for 10 minutes. The hot water was used to lyse the cells followed by chelex to help break away all of the debris that came out of the cells. The tube was then spun in a micro centrifuge for 90 seconds at 13,000 rpm. 30 µL of the clear supernatant was then transfer into a clean 1.5 mL tube and clearly labeled.
A Polymerase Chain Reaction (PCR) was then used to amplify the amount of DNA in the supernatant. The analyst obtained a PCR tube containing a Ready-To-Go PCR Bead and then labeled it with his/her initials. Using a micropipette with a fresh tip, 22.5 µl of PTC primer/loading dye mix was added to the tube, which allowed the bead to dissolve over one minute. Using a micropipette with a fresh tip, 2.5 µl of individual’s cheek DNA, prepared as stated above, was added directly into the primer/loading dye mix. Ensuring that there was no cheek cell DNA remained in the tip after pipetting. The analyst placed their PCR tube, along with other student samples, in a thermal cycler that had been programmed for 30 cycles of the following profile (figure 1). The profile was linked to a 4° C hold program after the 30 cycles were completed.
Denaturing step: 94 ° C
Annealing step: 64 ° C
Extending step: 72 ° C
30 seconds
45 seconds
45 seconds
Figure 1: 30 cycle Polymerase Chain Reaction Thermal Cycler Profile.
The analyst labeled a 1.5-mL tube with a “U” (undigested) and initials. Using a micropipette, 10 µL of PCR product was transferred to the “U” tube and stored on ice. 1 µl of restriction enzyme HaeIII was added directly into the PCR product remaining in the PCR tube

BTEC 1010, 12/08/13
Michael Turley and labeled with a “D” (digested). Reagents were mixed and pooled by gently agitating the bottom of the tube for 1 minute. The tube was then placed into a thermal cycler programed for
37° C for 30 minutes and stored on ice when complete.
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A 2% agarose solution was then poured into a gel tray with comb in place and allowed to solidify for 20 minutes (1 µl/mL ethidium bromide added to solution). The gel was then placed into the electrophoresis chamber followed by the addition of 1x TBE buffer solution until the gel was covered and the comb removed. With a fresh tip on a micropipette 20 µl pBR322/BstNI size markers was added to the far left Lane of the gel (figure 2). With a fresh tip on a micropipette 10 µl undigested “U” sample was added to lane 6. 16 µl of digested “D” sample was added to lane 7 (fig 2). The electrophoresis was run at 130 volts for 45 minutes, and viewed using trans illumination. An image of the results was then photographed.
The final phase of the experiment was to conduct a taste test to see if PTC could or could not be tasted. Analyst was given a plain strip of paper first as a control – we needed to know what the paper itself tasted like so we would not confuse the paper taste with the taste of PTC.
Then the strip of paper impregnated with PTC was tasted by each student to either confirm or dispute the electrophoresis gel results.
Results
Although the lanes 6 and 7 in the electrophoresis (figure 2) did not clearly show the digested DNA sizes (221 bp, 177 bp, 44 bp) relative to the example shown in figure 3, There was sufficient evidence that student 2 was heterozygote taster. Confirmation obtained when student 2 reported a mild taste of bitter after placing the strip of PTC paper on his tongue. Both the electrophoresis and the oral taste test indicated that the individual was heterozygote taster with the genotype Tt. (figures 1 and 2).
Ladder Student 1 Student 2 Student 3
Lane 1 lanes 3 & 4 lanes 6 & 7 lanes 9 & 10
177 bp
Figure 2: PTC genotype confirmation. Two bands confirm a heterozygote genotype (Tt). The tip of the arrowhead points to the first band. An additional band is seen directly beneath it.

BTEC 1010, 12/08/13
Michael Turley
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Figure3: Example of all three genotypes: dominant genotype (TT) or the heterozygous genotype (Tt). A non-taster would have the homozygous recessive genotype (tt). This example was used to help evaluate results.
Discussion
Analytical techniques utilized throughout this experiment allowed for interpretable data to determine the genotype of the collected DNA. The PCR used to amplify the DNA obtained from the cheek cells was highly thermo-stable as not to denature the protein contained in the
DNA. The DNA doubled in quantity once it was allowed to denature, anneal, and elongate, thus providing more DNA to be used in the ultimate interpretation of the genotype. A single band was identified on the gel after the initial separation and this result indicated that the DNA was amplified successfully. Further experimentation with precision should yield interpretable results that would allow an individual to determine his or her respective genotype.
The restriction digest performed used enzymes from the bacteria Haemophilus aegyptius.
HaeIII, which is a restriction enzyme that recognizes palindromes and is used to cleave DNA at specific sequence sites, cleaves DNA at the GGCC sequence site creating two fragments that end in GG. This particular sequence is found between three nucleotides of the amplified TAS2R38 gene, which means it is found only in PTC tasters. For a non-taster, the sequence would not be cleaved by HaeIII and would remain intact. The two separate bands observed in Figure 2 were able to confirm that the individual DNA tested is a heterozygote taster with the genotype Tt ability to differentiate among certain tastes may serve as an evolutionary adaptation. This adaptation would allow those with the PTC genotype to experience an array of bitter tastes not able to be experienced by non-tasters thus serving as a better defense mechanism against possible bitter toxins.