EXAMINATION OF THE TAS2R38 GENE AND ITS SPECIFIC NUCLEOTIDE DIFFERENTIATIONS TO DETERMINE ABILITY TO TASTE PHENYLTHIOCARBAMIDE
Although humans are essentially genetically identical as a whole, there are some minute variances in our gene coding that allow for differences in our interactions with the world. These genetic modifications may have extensive detrimental effects, small effects, or no apparent effect at all. A few of these alterations can even affect our senses. In this lab, we examine how a discovery by a scientist gives us insight into how a relative dissimilarity between humans can affect the ability or inability to taste certain chemicals.
Scientist Arthur ...view middle of the document...
This is done by restriction enzymes named restriction endonucleases that “chop” DNA at a certain region of a gene. One restriction endonuclease, called HaeIII, reads and cuts the DNA depending upon what nucleotide sequence is present at location 145 in the amplified 220 bp TAS2R38 gene region. Once the desired nucleotide sequence is recognized at this position (GGCG), this enzyme will clip the DNA at that region since there is a restriction site present at this allele, and will not clip if the DNA sequence is read as GGGG since there is no restriction site for “G” allele for HaeIII. This means that the PTC nontasting allele is recessive (t) and the PTC tasting allele is dominant (T). These slices of DNA fragments can be further examined by agarose gel electrophoresis which allows base pairs to be divided while using an electric current. Marker DNA is also useful, as it is used to approximate the size of the DNA fragments that have migrated through the agarose.
Since PTC tasting is a dominant trait (TT or Tt), certain techniques in the lab were used to figure out which alleles we possessed. We hypothesized that those students whose DNA bands were less than 220 bp had the “TT” or “Tt” genotype, and those whose bands were not cut had the “tt” genotype. We also hypothesized that those with the “tt” genotype would not be able to taste PTC (non-taster phenotype), while those with the “Tt” genotype would be mild tasters and those with the “TT” genotype would be strong tasters (tasting phenotype). In order to find out which alleles we possessed, we isolated DNA from our cheek cells and amplified the TAS2R38 gene which had an SNP. This was done by using a polymerase chain reaction (PCR), which first degraded template DNA by using a high temperature (95 degrees C). Then, the temperature was lowered (~50 – 65 degrees C) so that primers can attach to the single-stranded template DNA on the 5’ end to begin synthesis of a new strand by DNA polymerase. These repetitive steps allowed us to magnify our TAS2R38 gene sequence. Once amplified, our DNA sequence was recognized by the enzyme HaeIII and was cut (or not cut) dependent upon the base pairs found at this region. We set up our agarose gel electrophoresis using heated agarose powder in 1X TAE buffer, then allowed it to cool until it turned until a gel, with the DNA fragments being separated by wells. These DNA fragments were able to be separated due to an electric current being used to allow the negatively charged DNA to move toward the positive end of the electrode. In order to visualize the DNA, along with comparing them with marker DNA, ethidium bromide was added to the gel and running buffer to expose ultraviolet light by its binding specifically to double-stranded nucleic acids (Dolan DNA Learning Center 2006). We compared our digested by HaeIII and undigested DNA for us to confirm that the polymerase chain reaction was successful and used this to determine our genotypes.
In order to...