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In the 1850s, Charles Darwin introduced his groundbreaking theory of Evolution in his seminal work, "The Origin of Species." Darwin's theory posited that individuals within a population exhibit variations in their heritable traits. Furthermore, he suggested that if a specific trait confers a higher likelihood of fitness, individuals possessing that trait will tend to have more offspring than those lacking it (McAllister & Grewe, 2019).
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However, Darwin's theory did not elucidate the mechanism through which these traits were transmitted from parents to offspring.
Approximately 50 years later, Gregor Mendel conducted his pioneering experiments in plant breeding, shedding light on the mechanics of trait transmission.
Darwin's and Mendel's work collectively explained that genes, specifically the variations in alleles carried by individuals within a population, underlie the observable heritable traits and serve as the foundation of evolution. In the early 20th century, Godfrey Hardy and German physician Wilhelm Weinberg formulated the Hardy-Weinberg principle of equilibrium to describe the genetic makeup of populations.
The Hardy-Weinberg principle of equilibrium postulates the expected allele and genotype frequencies at a locus in a diploid, sexually reproducing population of infinite size in which mating is random (McAllister & Grewe, 2019). This principle relies on several conditions, including the absence of mutation, no selection among genotypes, no gene flow, infinite population size, and random mating. Gene flow occurs when genes transfer between populations through the migration of individuals, while random mating implies that the probability of two individuals in a population mating is uniform. Deviation from these conditions results in population evolution and genetic variation (McAllister & Grewe, 2019).
In this project, we employed Polymerase Chain Reaction (PCR) to investigate genetic variation at the LCT and TAS2R38 loci among students in the Foundation of Biology program.
The LCT gene, located on chromosome 15, encodes the lactase enzyme responsible for lactose digestion. Lactose, a disaccharide, is broken down into monosaccharides glucose and galactose by lactase in the small intestine's lumen. Epithelial cells in the small intestine absorb these monosaccharides and galactose. Individuals lacking the ability to produce lactase are considered lactose intolerant, a condition that naturally develops in approximately 60% of people as they mature into adulthood (Troelsen, 2005). The lactose-tolerant trait is indicated by alleles "TT" and "CT," while the lactose-intolerant trait is denoted as "CC."
The TAS2R38 gene, situated on chromosome 2, determines the ability to taste Phenylthiocarbamide (PTC). The PTC tasting ability has become one of the most studied human genetic traits. PTC is an artificial chemical synthesized by Arthur Fox in the 1930s. In addition to the two common alleles investigated in this lab, there are at least five rare alleles that affect the tasting phenotype (McAllister & Grewe, 2019). These common alleles include the dominant "T" allele for tasters and the recessive "t" allele for non-tasters.
Our hypothesis posited that the pooled data of individuals in the Foundation of Biology program would not adhere to the Hardy-Weinberg equilibrium for the alleles at the LCT and TAS2R38 loci. Our reasoning behind this hypothesis stemmed from the diverse ethnic backgrounds of the experimental group, suggesting gene flow within the parental generation. Additionally, we cannot assume that the parents of the students in the Foundation of Biology program mated randomly to produce this population. We concluded that the null hypothesis for this experiment was that the observed genotype frequencies within the Foundation of Biology class would conform to the expected frequencies for a population in Hardy-Weinberg equilibrium.
To examine the DNA sequences at the TAS2R38 and LCT loci, we employed PCR and subsequently used gel electrophoresis to identify individual genotypes for each target locus. PCR is a method for replicating a specific target sequence of DNA, generating billions of copies from a single or a few DNA templates (Laskowski et al., 2019). The PCR process necessitates a DNA template, DNA polymerase, deoxyribose-containing nucleotides, and primers that initiate synthesis at specific positions within the target DNA. In our study, we utilized restriction enzymes to initiate Restriction Fragment Length Polymorphism (RFLP) analysis. RFLP analysis allowed us to differentiate uncut DNA fragments from cut ones using gel electrophoresis. A Single Nucleotide Polymorphism (SNP) within the recognition site prevented the enzyme from cutting the allele sequence at that specific region, enabling us to distinguish between allele types.
Our primary objective in this project was to investigate genetic variation in the human population by collecting and analyzing epithelial cheek cells from 384 students enrolled in the Foundations of Biology class. We successfully genotyped each member of our group and aggregated class data to assess whether our class adhered to the expectations of Hardy-Weinberg equilibrium. To prepare for gel electrophoresis and genotyping, we employed Polymerase Chain Reaction (PCR) to amplify two target loci.
For the LCT locus, the forward primer was named LCT-F and had the sequence 5’- GTTGAATGCTCATACGACCATG-3’. The reverse primer, LCT-R, was designed with the sequence 5’- TGCTTTGGTTGAAGCGAAGATG-3’. In the case of the TAS2R38 locus, the forward primer, TAS-F, possessed the sequence 5’- AAC TGG CAG ATT AAA GAT CTC AAT TTA-3’, while the reverse primer, TAS-R, featured the sequence 5’- AAC ACA AAC CAT CAC CCC TAT TTT-3’ (McAllister & Grewe, 2019).
The PCR procedure commenced with the addition of an individual's DNA to a test tube along with Taq Polymerase, nucleotides, primers, and salts. Taq Polymerase, derived from a heat-loving bacterium found in hot springs, was utilized because it remains stable at high temperatures, unlike most cellular DNA polymerases. Primers, typically 20-25 nucleotides in length, are short synthetic DNA strands that are complementary to specific DNA sequences (McAllister & Grewe, 2019).
Subsequently, the test tubes were placed in a thermocycler, an instrument that amplifies DNA segments through controlled heating and cooling cycles. The thermocycler employed a three-step cycle comprising the denaturing step, annealing step, and polymerization step. In the denaturing step, the DNA was heated for five minutes at 95°C, followed by 40 cycles of 30 seconds at 95°C to separate the double-stranded DNA. In the annealing step, the reaction mixture was cooled to 55°C for 30 seconds, allowing DNA primers to attach to the template DNA. Finally, in the polymerization step, the temperature was raised to 68°C for 30 seconds to synthesize new DNA, followed by 5 minutes at 72°C to complete the synthesis (McAllister & Grewe, 2019). During this step, Taq polymerase generated a new DNA strand.
Following PCR amplification, we employed restriction enzymes for genotyping. Specifically, we used the BsmF1 enzyme for the LCT locus and the Fnu4H1 restriction enzyme for TAS2R38. In the case of the LCT gene, a heterozygous genotype "CT" exhibited three bands because the restriction enzyme cleaved the "T" allele into two bands while leaving the "C" allele uncut due to a Single Nucleotide Polymorphism (SNP) at the recognition site, resulting in a total of three bands (two cut and one uncut). The "TT" genotype displayed two cut bands as the restriction enzyme recognized and cleaved the "T" allele into two bands. Conversely, the "CC" genotype exhibited one uncut band as the restriction enzyme did not cleave at the recognition site due to the SNP in the "C" allele.
For the TAS2R38 gene, a heterozygous genotype "Tt" also displayed three bands. Here, the restriction enzyme cleaved the "T" allele into two bands while leaving the "t" allele uncut due to an SNP at the recognition site.
After the addition of restriction enzymes to the PCR products, the solutions were incubated in a heated water bath for 60 minutes. The DNA solution included Taq polymerase, nucleotides, primers, and salt to ensure proper polymerase function. Subsequently, the samples were pipetted into wells created in an agarose gel. We prepared a 1.6% agarose solution by mixing 0.64g of agarose with TBE buffer and ethidium bromide (40 mL). Agarose, a polysaccharide, separates DNA fragments based on their size. Ethidium bromide, a dye molecule, stains DNA fragments as they migrate through the gel, allowing us to track their movement. The DNA samples were mixed with a 10X loading dye and loaded into the wells. Gel electrophoresis was employed to separate DNA bands by size, taking approximately 40 minutes. Subsequently, we used a Fotodyne UV illuminator to capture an image of the separated DNA, which was arranged by base pair length (McAllister & Grewe, 2019).
We utilized the Hardy-Weinberg equations (p + q = 1 and p^2 + 2pq + q^2 = 1) and the Chi-square goodness of fit test (χ² = ∑[(o - e)² / e]) to analyze genotype and allele frequencies. These equations were essential in assessing whether we could reject the null hypothesis and analyzing how well our data conformed to the Hardy-Weinberg equilibrium. They provided insights into the genetic variation at the LCT and TAS2R38 loci within the Foundations of Biology class.
To assess genetic variation in the pooled class data, we analyzed the DNA fragments visible in the Fotodyne gel image. The gel data allowed us to determine individual genotypes for the two loci. The DNA size marker comprised five DNA fragments of known sizes (766, 500, 300, 150, 50 bp).
First, we encountered issues with the experiments of students 11 and 13. This problem arose because both students added the DNA size marker to their PCR DNA solution, resulting in the solution behaving like a DNA size marker. For the LCT loci in Figure 1, the enzyme BsmF1 recognized and cleaved the 'T' allele but did not recognize the "C" allele due to an SNP. Consequently, the "C" allele remained uncut, resulting in one DNA fragment at 386 bp. The "T" allele, with a BsmF1 recognition site, produced two bands at 238 and 148 bp. The heterozygote "CT" allele exhibited three bands at 386, 238, and 148 bp. For LCT loci in Figure 1, DNA sample #12 was identified as a homozygote "CC" allele, indicating lactose intolerance since the enzyme BSmf1 did not cleave the "C" allele. DNA sample #13 was identified as a heterozygote "CT" allele. DNA sample #14 was identified as a homozygote for the "TT" allele, indicating that the enzyme recognized and cleaved the "T" allele.
Next, the alleles for the TAS2R38 locus underwent Restriction Fragment Length Polymorphism (RFLP) analysis with the Fnu4H1 restriction enzyme. The "T" allele at position 785 of the PAV (taster) featured a recognition site at 5’- GCNGC- 3’, while the "t" (AVI) non-taster allele lacked this recognition site due to an SNP. Consequently, the "tt" allele, when cleaved by Fnu4H1, resulted in one band at 303 bp. The "TT" allele, recognized by Fnu4H1, produced two bands at 238 and 65 bp. The heterozygote "Tt" allele exhibited three bands at 303, 238, and 65 bp. For the TASR38 loci, DNA sample #11 and #12 were homozygotes "tt," indicating a non-tasting genotype. DNA sample #13 was a homozygote with a "TT" allele, indicating recognition of the "t" site and cleavage. DNA sample #14 was a heterozygote for the "Tt" allele.
| Genotype | Frequency |
|---|---|
| CC | #12 |
| CT | #13 |
| TT | #14 |
| Genotype | Frequency |
|---|---|
| tt | #11, #12 |
| TT | #13 |
| Tt | #14 |
We applied the Hardy-Weinberg equation to estimate allele and genotype frequencies for the loci under investigation. Comparisons were made between observed data and self-reported data. Additionally, we conducted the Chi-square test with 1 degree of freedom to evaluate the fit of the pooled data to Hardy-Weinberg expectations.
In the LCT loci, the obtained P-value was less than 0.0005, while for TASR38, the P-value was less than 0.001 but greater than 0.0005. Since both P-values were less than 0.05, we concluded that our class pooled data did not conform to the expectations of Hardy-Weinberg theory. Therefore, we rejected the null hypothesis.
The results of our study revealed deviations from the Hardy-Weinberg equilibrium expectations for both the LCT and TAS2R38 loci within the Foundation of Biology class. These deviations suggest that genetic variation exists among our sampled population, which may be attributed to factors such as gene flow, non-random mating, or other evolutionary forces.
For the LCT locus, the observed genotype frequencies significantly differed from the expected frequencies. In particular, the presence of individuals with homozygous "CC" alleles suggests lactose intolerance, as the BsmF1 enzyme did not cleave the "C" allele. This observation aligns with previous studies indicating that lactose intolerance is prevalent among certain populations (Troelsen, 2005). Furthermore, the identification of individuals with homozygous "TT" alleles demonstrates lactase persistence, confirming the presence of lactase-producing genotypes in our population. The occurrence of heterozygous "CT" genotypes further supports the notion of genetic diversity in our class. Overall, our findings at the LCT locus reflect the influence of genetic variation on the ability to digest lactose.
Regarding the TAS2R38 locus, our results showed a departure from Hardy-Weinberg equilibrium expectations as well. The presence of individuals with homozygous "tt" alleles indicates a non-tasting phenotype, consistent with previous research on the subject (McAllister & Grewe, 2019). Conversely, individuals with homozygous "TT" alleles exhibited a taster phenotype, confirming that they could detect the PTC compound. The presence of heterozygous "Tt" genotypes highlights the genetic diversity in our class concerning the ability to taste PTC.
While our results demonstrate genetic variation within our class, several factors may have contributed to these deviations from Hardy-Weinberg equilibrium. First, gene flow, which occurs when genes are transferred between populations through migration, could have introduced genetic diversity into our class. The diverse ethnic backgrounds of the student population may have played a role in this gene flow, leading to the observed deviations.
Second, non-random mating within our class may have influenced the genetic composition. It is unlikely that the parents of the students in the Foundation of Biology program mated randomly, which could result in deviations from the Hardy-Weinberg equilibrium. Non-random mating based on genotype or other factors could contribute to the genetic variation we observed.
Lastly, selection pressure could have influenced the genetic makeup of our class. If certain genotypes provide a selective advantage, they may become more prevalent in the population. This selective pressure could result in deviations from the expected genotype frequencies under Hardy-Weinberg equilibrium.
In conclusion, our study aimed to investigate genetic variation at the LCT and TAS2R38 loci within the Foundation of Biology class. Our results indicate that the genetic makeup of our class does not conform to the expectations of Hardy-Weinberg equilibrium for these loci. The presence of lactose intolerance and lactase persistence genotypes at the LCT locus, as well as the variation in PTC tasting abilities at the TAS2R38 locus, highlight the genetic diversity within our population.
Our findings suggest that gene flow, non-random mating, or selective pressures may have influenced the genetic composition of our class. These factors, combined with the diverse backgrounds of our students, likely contributed to the observed genetic variation. Our study underscores the importance of considering genetic diversity when examining human populations and their genetic traits.
Further research and a larger sample size may provide more insights into the genetic variation and its underlying causes within the Foundation of Biology class. Understanding the dynamics of genetic variation can shed light on the evolutionary forces shaping human populations.
Genetic Variation in LCT and TAS2R38 Loci. (2024, Jan 23). Retrieved from https://studymoose.com/document/genetic-variation-in-lct-and-tas2r38-loci
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