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The key goals in performing this study in-lab was to determine how PCR can be used to amplify desirable segments of DNA, examine how agarose gel electrophoresis can separate DNA fragments via size, and analyze DNA fragments on agarose gels to determine our PTC diplotypes. The collection of this data was done by gathering a sample of cheek cell DNA, then combining said cells with an InstaGene™ matrix (BioRad Laboratories) to isolate the cheek cell DNA (Lintott 4). It was necessary to use a chelating agent, such as the InstaGene™ matrix, to chelate the metal ions contained within the solution during the lysis step when the tube containing the matrix was placed on the 100℃ heating block (Lintott 5). The matrix was able to remove metal ions responsible for degrading DNA; therefore, the DNA template was able to remain intact. Subsequently, a master mix was added to each PCR tube for gel electrophoresis, which is essential for amplifying the DNA templates (Lintott 19). The DNA fragments could then be examined to determine which bands were larger (slower moving) and which were smaller (faster moving). This step was essential for determining our PTC diplotypes. Additionally, bioinformatics research was critical to understand our results.
Based on the PTC taste test and information gathered through bioinformatics analysis, I predicted that I was a heterozygous non-taster. I found the PTC strips to have little bitter taste, and knowledge acquired via bioinformatics analysis of the taster (AY258597) and non-taster (TAS2R38) genes further supported my prediction. This prediction proved to be true when our DNA samples were run on a gel and DNA fragment sizes were examined. Using modern DNA technology, our diplotypes could be determined. The impact of DNA technology on society today has several advantages and disadvantages. Some benefits of personal genomics includes diagnosis of threatening but treatable conditions, carrier screening for inherited disorders, pharmacogenomics, and discovering links to ancestry. Additionally, these benefits can improve overall health to those individuals who wish to become aware of potential health conditions or factors. Some disadvantages of sequencing genomic DNA include misuse or abuse of data, insufficient support and guidance from health care providers to patients, inadequate interpretation of data, and ethical challenges such as informing the patient that they possess a lethal condition.
There are three SNPs found in the TAS2R38 gene which is associated with the capacity to taste PTC, resulting in two alleles: PAV (taster) and AVI (non-taster) allele. Table 1 depicts the polymorphisms within the gene. This experiment was able to identify actual diplotypes at each of the SNP positions. Individuals who are homozygous tasters possess the PAV/PAV diplotype, homozygous non-tasters possess the AVI/AVI diplotype, and heterozygous individuals possess the PAV/AVI diplotype, all of which can be determined by analyzing the DNA fragment lengths on the agarose gel. After examining the fragment lengths of DNA and determining the band sizes, we further analyzed our data by using the chi-squared test. The chi-squared test is a statistical hypothesis test that can be used to test the relationship between categorical variables to determine if there were any correlating factors resulting in some individuals possessing vs. not possessing bitter taste receptors. The p-value was also calculated to determine the certainty of the chi-squared results. Using R and RStudio, we examined the relationship between several categories, including phenotype and having a preference for the colour red (PrefRed), phenotype and wearing green (WearGreen), phenotype and designated Harry Potter Hogwarts house (HP), phenotype and being a smoker, genotype and PrefRed, genotype and WearGreen, genotype and HP, and genotype and being a smoker. It was calculated that none of the variables (PrefRed, WearGreen, HP, smoker) had an effect on phenotype or genotype.
This lab was very beneficial and relevant to lecture material. Not only were we able to discover how PCR may be used to amplify segments of DNA, but additionally, we could examine DNA fragment size and determine our PTC diplotypes via agarose gel electrophoresis. This concept, as well as other modern-day DNA sequencing methods, are advantageous for determining overall health and fitness. However, modern-day DNA sequencing may also be disadvantageous, and could result in the misuse or abuse of data. By analyzing the data and DNA fragment band sizes, we were able to determine our diplotypes and further test the relationships between categorical variables using the chi-squared test; therefore, we could dictate correlating elements between possessing vs. not possessing the PTC tasting gene.
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