Acheampong Wiredu Final Lab Report: PTC.
Genetics bio2450
05/11/19
Abstract
Polymerase chain reaction (PCR) is the technique this laboratory experiment focusses on for an excellent result. The reason behind the used of PCR is to raise a specific gene of DNA, which in this experiment the use of a piece of saturated phenylthiocarbamide (PTC) paper specify put in the mouth to extract the DNA from the cheek cell to confirmed taster or non-taster. The agarose gel electrophoresis is used to confirm if the PCR product reaction worked. Haelll enzyme determines the PTC genotype, which the taster would indicate the possible genotype of homozygous dominant (TT) or heterozygous (Tt) and the non-taster would have just one possibility genotype of homozygous recessive (tt). Genotype is the results of the band(s) located on agarose gel that could be visualize through the ultraviolet light. The single band above the position of the reference ladder is called a homozygous recessive (tt) meaning non taster, single band below the reference ladder is called a homozygous dominant genotype (TT) meaning taster and two bands is called a heterozygous (Tt) also meaning taster.
Introduction:
In this laboratory experiment, it is important to pay attention on genetics taste receptor discovered on the tongues of vertebrates. There are different tastes when food gets contact with the tongue for the first time and these sensations are sour, sweet, salty, bitter and savory. These sensory tastes are varied in individual of receptors that bind to the molecules that result in our perception of these sensations. The phenylthiocarbamide (PTC) is the receptor senses presence chemical when individual tastes bitter. The PTC ability of taste is generated from the gene called TAS2R38, which encodes a protein to our tongues that communicate the bitterness of this chemical. In 1931, an American chemist Dr A. L. Fox discovered the taste of phenylthiocarbamide by preparing a quantity of phenylthiocarbamide and transferred this compound in a bottle. During the transferring, some phenylthiocarbamide accidentally diffuses into the air in his laboratory. He noticed that another chemist standing not far away could taste the bitterness, but he tasted nothing. This surprised Fox discovered the ability to taste phenylthiocarbamide by being closer is to inhaled more but apprehend on taste. His tasteless took him so far to taste crystals and found them tasteless too. Whiles other chemist was able to taste the bitterness of the crystal. The substance was declared by some people as tasteless and others bitter. PTC is one of studied experiment on human taste, which proves that close to 75 percent of people taste as extremely bitter and remaining 25 percent of people as barely tasters. (Fox et al. 2009). There are two allele of TAS2R38 gene with minimum five more rare variants. A single nucleotide polymorphism (SNP) is supervise the replacing of one amino acid in the receptor. The difference of amino acid changes the ability of the receptor to respond or not respond to PTC. Our gene are received from both our parents based on where the gametes are formed and what alleles distributed during fertilization. The gender, age and environment of humans contributes a lot when it comes to PTC (tasting molecule). Females have a high sense of tasting a molecules substance than males. The atmosphere of the environment also affects our sensibility of tasting molecular substances. An example is the experiment contact with the growthand development from infancy to age 18 years in tasters and non-tasters of phenylthiocarbamide (P.T.C.) were studied between 50 female and 44 male subjects, which girls that were able to taste reaches maturation have an average of 3·8 months earlier than non-taster girls. The reverse was true for boys, non-tasters maturing on average 6·2 months earlier than tasters. There is effect of age on taste sensitivity. An experiment used to test 100 subjects with the age between 15 to 89 for taste sensitivity to substances of a sour, salty, sweet, and bitter nature. At the conclusion of this experiment, was found that the sour, salty, sweet and bitter nature between the ages were essentially same.
Methods:
The used of sterile cytobrush was insert into the mouth to extract fragment of DNA from saliva. This process is done by leaving the cytobrush inside the mouth for 25 times to attained cheek cells. The brush is then put into a microcentrifuge tube ofChelex suspension (100 ul, 10% w/v). The tube is placed on 100 °C heat block for 10 minutes and spun in a centrifuge for 5 minutes. After being centrifuge and suspension, the debris from the cells formed a pellet at the bottom of the tube and the DNA was in the supernatant. The supernatant is move to clean microcentrifuge tube and stored at -20 °C. for a 7days. A polymerase chain reaction (PCR) is used to amplify the amount of DNA in the supernatant obtained from the previous week. A piece of PCR in tubes is obtained, experimental and negative controls were place in different tubes. In the experimental tube,22 μl of primer mix is added to PCR tubes with PCR beads. The forward primer 5’-CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG-3’and reverse primers 5′-AGGTTGGCTTGGTTTGCAATCATC-3′ were deleted so as not amplify the PTC gene, but to permitted unspecified amplification of a product. Denaturing process is done by heating the DNA at 95 °C to denature the DNA from a double stranded molecule to a single stranded molecule. Annealing is the next stage by setting the tube to 62 °C cooled and the primer to target the DNA. Final stage is elongation, which the tube is then heated to 72 °C for new DNA strands elongated. The restriction enzyme digestion follow by gel electrophoresis are next. The goal is to focus on the setup restriction digests on the PCR products to express the allelic polymorphisms. Recall that the recognition site for the HaeIII enzyme is GG|CC. In other words, the enzyme will only cut the DNA at these sequences.The agarose gel prepared in a flask with TBE buffer with 1g agarose. The solution is mix and heated inside microwave for a completely dissolved buffer. Addition of SYBR Safe DNA stain to the hot flask to mix and set two combs into each gel apparatus. Carefully transferred the gel into the gel tray without no liquid is spilled and placed in refrigerator to precipitate solidification. When DNA dissolution, get two new microcentrifuge tubes with letter ‘U’ for undigested and the letter ‘D’ for digested. Transfer 5 ul of PCR product into tube labeled ‘U’ and 5 ul into tube labeled ‘D’ and add 1 ul of HaeIII enzyme to tube ‘D’. Pipette the mixture to homogenate and incubate for 10 min at 37 °C and save the remaining 15 ul of PCR product for DNA sequencing.Use gel electrophoresis to separate and visualize the DNA fragments resulting from digestion with the HaeIII restriction enzyme and the goal is to compare the observed genotypes among individuals. When solidified, tranfer gel in gel apparatus and set up the electrode. Loaded two samples into the gel, one undigested sample and one digested with the restriction enzyme. Each gel should also include a DNA ladder (marker) to help size the fragments (load 5 ul of the DNA ladder). Run gel for 20 min at 120 V and visualize results using transilluminator and obtain genotypes
Results:
The result on the agarose gel electrophoresis indicate the presence of PCR product shows that I am a strong taster due to the indication of the two bands on the gel electrophoresis which established that there was a presence of a dominant allele in the genotype. Since I was able to taste it, my DNA tested is a heterozygote taster with the genotyope Tt. A heterozygote can be explained as an individual who possess two different alleles of the same gene. This indicates that my mother is either a homozygous dominant taster (TT) or heterozygous taster (Tt) and my father is a homozygous recessive taster (tt).
Discussion:
In conclusion of this laboratory experiment, there were some multiples of error generate during the time of getting the DNA, inexact time lapse in between steps, error with the process of PCR and agarose gel electrophoresis. These errors can be avoided by getting more DNA during extraction and timing should be considered in each step and performing PCR and preparing gel electrophoresis should be done with cautioned. All the procedure performed in this experiment with gene could be analyzed to determine genotypes for specific phenotype. In the field of forensics whereby DNA laboratories could use the genotype obtained for a specific trait by individual to use as evidence in crime scene investigations. This laboratory experiment is helpful and benefit in the exoneration or persecution of an individual in question.
Reference List:
Wooding, Stephen et. al. “Natural Selection and Molecular Evolution in PTC, a Bitter-Taste Receptor Gene.” American Journal of Human Genetics (2004) 74:637-646, accessed October 24, 2010.
Fox, A. L., 1932 The relationship between chemical constitution and taste. Proc. Natl. Acad. Sci. USA 18:115-120
Cooper, R. M., Bilash, I., & Zubek, J. P. (1959). The effect of age on taste sensitivity. Journal of Gerontology, 14, 56-58.
AKCASU, A., and OZALP, E., 1977, Distribution of taste thresholds for phenylthiocarbamide among different age groups in Turkey. Pahlavi Medical Journal, 8, 294–304.
ANLIKER,J. A., BARTOSHUK,L., FERRIS, A. M., and HOOKS, L. D., 1991, Children’s food preferences and genetic sensitivity to the bitter taste of 6-n-propylthiouracil (PROP). American Journal of Clinical Nutrition, 54, 316–320.
