Introduction:
To determine how genotype determines phenotype, genetic sequence analysis is commonly used — which, in turn, helps us to better understand human biology as a whole. A pivotal role of genetic variation: what distinguishes our species from other species is simply our ability to distinguish — how we think, how we act and react, and how we grow and develop as independent individuals in society. We can attribute much of these differences to our senses — sight, smell, hearing, touch, and finally, taste. Humans perceive toxins and nutrients qualitatively and automatically — as sweet, savory, or bitter tasting. The most commonly studied, and also the most divisive, bitter compound is phenylthiocarbamide (PTC) and how it affects us. The ultimate goal of this experiment was to determine each student’s personal allelic genotype and predict their ability to taste PTC. Variations of this experiment have been conducted multiple times — all serving to portray the phenomenon of human evolution.
With the experiment performed by Stephen Wooding et al., it was determined that chimpanzees developed the PTC taste sensitivity alleles independently of humans 1, which is proof of convergent evolution. Also, through the results of an experiment performed by Un-kyung Kim et al., they pinpointed the location of the PTC tasting gene — the TAS2R38 loci 2. These genotypes also might’ve been concentrated around a general region proven by Michael C. Campbell et al. The results of his experiment showed striking patterns of variation at TAS2R38. But these patterns also showed rare polymorphisms focused only in Africa. These high frequencies in Africa were of haplotypes associated with bitter taste sensitivity, and were found across genetically and culturally distinct Africans 3.
Additionally, a study by Carles Lalueza-Fox et al., outlined how PTC sensitivity has been prevalent in humans virtually since Neanderthals walked the earth. This specific finding revealed that not only were Neanderthals able to taste PTC, but they were heterozygous in their ability to do so. This shows the evolutionary importance and indicates a pattern. PTC-tasting ability was crucial for survival — they needed this ability to detect toxins or other harmful presence in foods 4. Between these four experiments, they all showed that PTC is a topic of interest among many fields — in both evolutionary history and biological needs. Knowing this, we ran an experiment to isolate this specific TAS2R38 locus.
All in all, the tasting of PTC is acknowledged as something of a divisive nature — some people can taste its bitterness while others taste nothing at all. We treated the class as a population, and we used data to further determine the allele frequency, and see if the population was truly in a Hardy Weinberg equilibrium. To harvest this data, there were many factors involved —purification of each students genomic DNA, amplification of the PTC locus using PCR, performing a restriction digest on the PCR product followed by gel electrophoreses to visualize the PTC allelic variability, and to then, perform Hardy-Weinberg analysis on genotypic and phenotypic data together with classmate data. What was surprising what that even though the data of each specific class section varied, the population as a whole stayed constantly, unwaveringly in Hardy-Weinberg equilibrium. This would mean that natural selection — the differential survival and reproduction of individuals due to differences in phenotype — did not have an impact on our population. But, one abnormality stood out as we were analyzing our data: some genotypes didn’t match their phenotypes. Some students had the genotype of a heterozygous taster and were still unable to detect the PTC. Another factor must’ve been at play, which could’ve either been an additional mutation or another underlying issue.
Materials and Methods:
As mentioned earlier, we first purified the DNA samples by rinsing our mouths with a saline solution. We transferred this saline into a microfuge tube and spun it in a balanced centrifuge at full speed for two minutes. Then, disposed of the supernatant, and added 400μL of lysis solution to the tube. We placed the tube into a 56ºC thermoblock and incubated for 15 minutes. At the halfway point (about 7-8 minutes), we removed the tube, shook them gently and placed it back in the thermoblock. After this, we put it back in the centrifuge for 5 minutes, removed extra ethanol with a micropipette, left the tube open for about 5 minutes, and finally, added 200μL of TE buffer (contains Tris buffer and EDTA) to the DNA pellet.
Our samples were now ready for PCR. We added 10 μL of 2X GoTaq green master mix, 2.0 μL of DNA, 4.0 μL of 5 μM PTC forward primer, and 4.0 μL of 5 μM PTC reverse primer to the PCR microfuge tubes. We mixed them gently, placed the tubes on ice, and put them through the general PCR process – 1 cycle of 94˚C for 60 seconds, then 35 cycles of 94˚C for 30 seconds, 54˚C for 45 seconds, and 72˚C for 45 seconds.
We then created our restriction digest mix by taking our PCR samples, and adding 4 μL of the PCR and 8μL of Fnu4H1 Restriction enzyme to the microfuge tube. We then closed the tubes gently and incubated the restriction digest at 37°C overnight. At the next session, we proceeded with agarose gel electrophoresis. One member of the lab loaded a 5μL of molecular weight marker into the first lane of the gel. Next, we added 10μL of the restricted PCR reactions to one of the empty lanes on the gel. Once everyone had loaded his or her sample, the gel ran for 30 minutes. At the end of the run, a picture of the gel was taken using a computerized transilluminator 5.
Results:
The purpose of this experiment was to demonstrate the relationship between phenotype and genotype. With a PTC paper strip, we determined which students were tasters or which were non-tasters. Using the results from the gel, we determined if phenotype matched genotype. We also used the Hardy-Weinberg equation on the PTC data to calculate the frequencies of the taster vs. non-taster alleles in the population. After calculating, we found that the allele frequencies equaled 1, and was indeed in Hardy-Weinberg Equilibrium.
Table 1
Figure 1
Hardy-Weinberg Calculations 6
In our class we had:
9 Non Tasters (qq).
5 Heterozygous Tasters (pq).
3 Homozygous Tasters (qq).
This was a total of 34 alleles in our population.
p+q=1. (23/34)+ (11/34) = 1
q=23/34=.676 –> .676^2 = .457
p=11/34=.323 –> .323^2 = .104
p x q = .218 –> 2pq = .437
q^2 + p^2 + 2pq = 1
.457 + .104 + .437 = .999
As shown in Table 1, which was a quantitative review of all class sections, the majority of the students were heterozygous, with an 86.5% match rate. However, homozygous dominant individuals had an 87.5% match rate and homozygous recessive individuals had an 84.4% match rate. These calculations were found by dividing the number of genotypes from the observed phenotypic results. The resulting percentage served as the phenotype-genotype match rate. Figure 1 was a photo of one of our gels after the gel electrophoresis process. This was after our gel was purified with a lysis buffer, amplified with PCR, and digested with a restriction enzyme. The majority of the gel shown was also homozygous dominant.
Discussion:
The average match rate of 86.13% supports our hypothesis — that after all, there is a strong, undeniable correlation between phenotype and genotype. PTC analysis is simply a method to provide more information about our genetic heredity and has been a focus of many other scientific pursuits — including the works of Stephen Wooding, Un-kyung Kim, Michael C. Campbell, and Carles Lalueza-Fox. These discoveries further help us predict outcomes to certain diseases and mutations — helping mankind as a whole. Our results followed in the same pattern. Only less than 20% of our data did not match, but this simply could’ve been due to the failure of proper taste perception, even from something as simple as what that student had eaten right before the taste test. Further trials should’ve followed to detect any other underlying reasons, and to help answer the unpredictable phenomenon of non-matching. But due to the over 80% of our results, we can look at the larger picture and decide confidently that the connection between genotype and phenotype is undeniable, even between the over 100 students that participated in this study. Works Cited
Stephen Wooding, Un-kyung Kim, Michael J. Bamshad, Jennifer Larsen, Lynn B. Jorde, and Dennis Drayna
Wooding, Stephen et al. “Natural Selection and Molecular Evolution in PTC, a Bitter-Taste Receptor Gene.” American Journal of Human Genetics 74.4 (2004): 637–646. Print.
Kim, U. K., Jorgenson, E., Coon, H., Leppert, M., Risch, N., Drayna, D.
Kim, et al. “Positional Cloning of the Human Quantitative Trait Locus Underlying Taste Sensitivity to Phenylthiocarbamide.” Science, American Association for the Advancement of Science, 21 Feb. 2003.
Michael C. Campbell, Alessia Ranciaro, Alain Froment, Jibril Hirbo, Sabah Omar, Jean-Marie Bodo, Thomas Nyambo, Godfrey Lema, Daniel Zinshteyn, Dennis Drayn
Campbell, Michael C. et al. “Evolution of Functionally Diverse Alleles Associated with PTC Bitter Taste Sensitivity in Africa.” Molecular Biology and Evolution 29.4 (2012): 1141-1153. PMC. Web. 24 Apr. 2018.
Carles Lalueza-Fox, Elena Gigli, Marco de la Rasilla, Javier Fortea, and Antonio Rosas
Lalueza-Fox, Carles et al. “Bitter Taste Perception in Neanderthals through the Analysis of the TAS2R38 Gene.” Biology Letters 5.6 (2009): 809–811. PMC. Web. 24 Apr. 2018.
Joseph Osmundson
Osmundson, Joseph. From Genomes to Biomes: Principle of Biology Laboratory Manual. New York University.
Calculations provided by Stephanie Banakis.