Biomolecular Techniques in the Taste Perception of Phenylthiocarbamide - Research Paper Example

Biomolecular Techniques (Bitter Taste Perception Of Phenylthiocarbamide (PTC) By Presented to Due Date ABSTRACT The ability of an individual to taste PTC depends primarily on their genetic makeup and is controlled by the PTC gene known as the TASR238 taste receptor gene, located on the chromosome 7 (7q34) and is about 1003 bp long. PTC sensitivity is a Mendelian trait where non-tasters possess two recessive alleles (tt) while the tasters have a dominant allele (T); and hence this genetic basis has been used in tracing populations migration and family lineages. The ability to taste PTC is an inherited, and hence populations from a single race will have a similar percentage of tasters and non-tasters. The project was done to determine the phenotypic and genotypic characteristics of students, and the results that were obtained were matched to those of European and Sub-Saharan cohorts. It was found allele combination of homozygous tasters, heterozygous tasters, and homozygous non-tasters were similar to those of the European cohort. This implied that the experiment was largely successful and accurate for the determination of phenotypes and genotypes of the PTC gene. The results can be used in making informed decisions with regard to dietary intake of foods rich in anti-oxidants, in planning of alternative nutrient rich meals for children that are sensitive tasters and finally it can be used by clinicians in the treatment plan of cancer or cardiovascular complications patients. 1.0 Introduction Every individual is different from another, and this is attributed to their genetic make-up. The sense of taste also varies between different categories of people where, for example, some people can sense the taste of some chemicals while others are not able to. One such chemical is phenylthiocarbamide (PTC) or phenylthiourea an organic compound that tastes very bitter to some people while others are unable to taste it at all. Studies done in the past have indicated that polymorphisms in sensory receptor genes in humans can alter the perception in individuals through the coding for receptor types that are functionally distinct (Bufe et al., 2005). The ability of an individual to taste PTC depends primarily on their genetic makeup and is controlled by the PTC gene known as the TASR238 taste receptor gene, located on the chromosome 7 (7q34) and is about 1003 bp long. There are three coding SNPs that are non-synonymous within the taster TAS2R38 gene which are: rs713598–G145C, Ala49Pro; rs1726866–T785C, Val262Ala; rs10246939 – A886G, Ile296Val, and are responsible several haplotypes (Kim, et al 2005; Bufe , 2005). PTC sensitivity is a Mendelian trait where non-tasters possess two recessive alleles (tt) while the tasters have a dominant allele (T), and hence this genetic basis has been used in tracing populations migration and family lineages (Ooi et al., 2010; Moberg et al.,2007). Kim et al. has found that the principal genetic determinant for such phenotypic variations exists within the population in PTC taste sensitivity are alleles of the TAS2R38 gene; a gene that located on chromosome 7 (Dotson,2010). PTC/PROP bitter taste receptiveness at locus TAS2R38 is a well-established guide of individual alteration in the oral sensation that has been related to predicting food taste and consumption (Hauxwell, 2013). Persons who possess enhanced the perception of PROP may avoid eating foods that are bitter-tasting including vegetables such as broccoli and some fruits (Moberg et al.,2007). A person’s phenotype indicates whether he/ she is a taster, mild taster or a non-taster and can be determined easily. An analysis of the genotype requires an understanding of an individual’s genetic make- up of at the molecular level where the allele types are determined, a more complex process. The Purpose of this project was to determine the phenotypic as well as the genotypic character for the PTC gene amongst students and then compare the results of both analyzes. The two PTC alleles differ at locations in the PTC gene nucleotide positions 145, 785 and 886. By using a suitable restriction enzyme, it is possible to cut off the dominant allele at a specific site and then identify the genotype of an individual with the taste PTC receptor gene. The SNP at position 785 is significant in the recognition of the genotype. For an allele that is dominant a restriction enzyme such as Fnu4H1 is used to recognize the restriction site located at C785 and proceeds to cut at that point. This site is lacking in a non-taster with recessive alleles where C is lacking, and T is present. PCR amplification gives clear results that indicate the tasters and the non-taster individuals. METHODOLOGY A sterilized wooden splint was used for preparing the buccal cells, a vertex, a varied temperature heating block, thermal cycler, centrifuging, micro-Eppendorf tubes, 1.5 ml Eppendorf tubes, UV trans-illumination, and electrophoresis were the materials used in carrying out the experiment. In the first week, the sample was extracted using a sterilized wooden splint to remove buccal cells that were transferred into a 1.5 ml Eppendorf tube that contained 350µl Chelex suspension. This was done using a using a sterilized plastic loop. An f 4 µl of Proteinase K solution was then added to the tube. The tube that now contained a mixture of these components was incubated for 30 minutes at 56°.It was then vortexed for 10 seconds and later centrifuged for 20 seconds at 13,000 rpm. A volume of 3.5 µl master matrix that was constituted by the ideal PCR components for optimal detection of the PTC gene and 6.5µl of the template DNA buccal cell were transferred into another micro-Eppendorf tube. The sample was dissolved and, the tube placed on the thermal cycler for amplification of the DNA fragments. The solution was then heated as follows: 94˚C 4 minutes 55˚C 40 seconds 72˚C 40 seconds 40 cycles 94˚C 40 seconds 55˚C 5 minutes 72˚C 5 minutes In the second week, Restriction Endonuclease enzyme, 5µl, was added to a tube that contained 10µl of the master mix and template DNA that had been prepared in the previous week. The tube was placed in a heating block at37°C. In the third week, loading buffer, 5µl, was added to a tube that contained 20µl of PCR/R Enzyme Digest residual and then mixed thoroughly. 10µl of PCR product, as well as a 100bp ladder marker lane, were loaded into a two%agarose gel well. The samples were subjected to electrophoresis for 60 minutes at 7cm-1. The gel was then stained using 0.5µl ml-1 ethidium bromide and destained for 5 minutes under running tap water. This was then photographed under the UV transillumination for differentiation of bands. A total of 216 students participated in reporting PTC sensitivity, and the results compare to the Sub-Saharan and European cohorts that were obtained from PubMed. Results The electrophesis results show that I am a mild taster heterozygous Tt hence three faint bands were formed. Distance traveled (mm) Ladder fragment (Log) 11 3 12 2.954242509 13 2.903089987 14 2.84509804 16 2.77815125 18 2.698970004 20 2.602059991 23 2.477121255 27 2.301029996 32 2 Fig.2. Representing Graph plotted from the data obtained from the Log (MWT) against distance traveled by the ladder fragments (data are on the table). Calculation: From the graph the equation is y = -0.0458x + 3.5083 In which Y= Molecular Weight fragment, X= Traveled Distance Unknown MW of undigested DNA as measured by ruler ( 23 mm) y = -0.0458x + 3.5083 y = -0.0458(23) + 3.5083 y = -1.0534 + 3.5083 y = 2.4549 y = anti log 2.4549 y= 285.036 Unknown MW of Digested DNA as measured by a ruler (25 mm) y = -0.0458x + 3.5083 y = -0.0458(25) + 3.5083 y = -1.145+ 3.5083 y = 2.3633 y = anti log 2.3633 y= 230.834 Table 1: Allele frequency of student’s class Allele type C T Number (216) 93 123 Allele frequency 0.430 (43%) 0.569 (57%) Table 1: the percentage and the frequency of 108 students who are either a homozygous or heterozygous Genotype taster genotype (CC) mild tasters (CT) non-tasters (TT) Number of persons 44 106 76 Frequency 0.195 0.469 0.336 Frequency as percentage 20 50 30 Allele frequency of European people Allele type T C Number (452) 258 194 Allele frequency 0.571 0.429 Percentage and the frequency of 224 persons who are either a homozygous or heterozygous (sub-Sahara cohort) Allele frequency of sub-Sahara people Allele type C T Number (448) 306 142 Allele frequency 0.683 (68.3%) 0.317 (31.7%) Chi Square from Mini tab: Student Vs European Rows: Worksheet rows Columns: Worksheet Columns C785 T785 All 1 93 123 216 92.8 123.2 2 194 258 452 194.2 257.8 All 287 381 668 Cell Contents: Count Expected Count Pearson Chi-Square = 0.001; DF = 1; P-Value = 0.974 Likelihood Ratio Chi-Square = 0.001; DF = 1; P-Value = 0.974 Student Vs Sub Saharan Africa Rows: Worksheet rows Columns: Worksheet Columns C785 T785 All 1 93 123 216 129.8 86.2 2 306 142 448 269.2 178.8 All 399 265 664 Cell Contents: Count Expected Count Pearson Chi-Square = 38.738; DF = 1; P-Value = 0.000 Likelihood Ratio Chi-Square = 38.405; DF = 1; P-Value = 0.000 Discussion Perception of the bitter taste is attributed to the detection by a wide range of G protein-coupled receptors (GPCRs) that encompass more than 25 diverse sequences which enable them recognize an equally diverse chemical moieties (Dotson et al, 2010; Greene et al,2011) hence an individual’s ability to taste PTC is determined by the gene TAS2R38 gene (Meritt et al., 2008). The analysis of this PTC gene is of significant as TAS2R38 is attributed to different people’s eating behaviors amongst other human phenotypes. (Goldstein et al.,2000; Inoue et al. 2013; 2000; Wooding et al. 2006) Of the entire PTC genes that were sequenced, a BLAST search was done from the data collected. Analysis using electrophorectic gel showed that my sample was indicative of a mild taster with heterozygous taster alleles. Comparison of the sequence with both a taster and non-taster allele following two BLAST searches showed that the results from a 98.7 % query with the taster allele and 98.8% query result against a non-taster allele was observed. The E-values for both the taster and non-taster allele was close indicating that the alleles from my sample were similar to those of a taster as well as those of a non-taster hence indicating that I am a heterozygous taster with both dominant and recessive taster genes. The class population samples presented all three phenotypes: TT, Tt and tt that implies the t unequal allele distribution within a given population. It was noted that most of the students were tasters the question that need to be determined was whether they were homozygous or heterozygous tasters. The Chi Square probability with the Sub-Saharan and European results showed that there was a higher probability of student results matching those of the European population with the P value being 0.974. The findings from the class population closely matched those of the European cohort implying that most of the students had inherited this gene. The two haplotypes vary due to the three SNPs that are responsible for the functional alleles production referred to as the PAV(Proline, Alanine and Valine) in tasters and AVI ( Alanine, Valine and Isoleucine ) in non-tasters. The statistics from the data collected in the class rejects the null hypothesis showing no difference between the two values with no chance of a difference arising. The data collected from the class had a close correlation to that of the European population. This shows that the experiment carried out in the university laboratory was accurate and that the samples, equipment and method used were in line with the standard procedures for testing PTC phenotypes and genotypes. The study from the class revealed similar results to those done across the globe that shows that the majority of the world’s population comprises of tasters (5, 18, 19). In addition, the results that were obtained from the analysis using gel electrophoresis showed that DNA separation is based on the genotype of the sample where tasters produced two bands, non-tasters produced one band while mild tasters produce three bands. ( Shivaprasad et al., 2012; Lee et al.,2012). The TAS2R38 gene SNP at position 785 is of significance as it determines the PTC genotype as the sequence in tasters has a restriction site that replaces C785 with T785 when subjected to a restriction enzyme. This restriction site forms a is a specific sequence that the restriction enzyme recognizes and cleaves to and results in the formation of multiple bands in taster samples analyzed by gel electrophoresis and a single band in non-tasters as the enzyme does not cleave to any site. Some results were not in line with the student phenotypes; some students reported having the ability to taste the PTC while the results indicated otherwise. This shows the subjective nature of the experiment resulting in data misinterpretation. This experiment is useful in its findings as it explains the variance in food preference that is observed amongst given populations; in this case the class population. People that are homozygotes PAV/PAV have a very high sensitivity to PTC as compared to the heterozygotes PAV/AVI) tasters or the homozygote AVI/AVI non-tasers. The allele presence affects eating behaviors where persons with high sensitivity to the bitter taste tend to dislike vegetables such as broccoli, as well as other bitter fruits. (Dotson,2010). This can influence a person’s diet where preference may be for sweeter or bitter foods and hence affects BMI of individuals (Goldstein,2000). The findings of this experiment can by clinicians and dietitians when planning a patient’s diet. A diet that is rich in glucosinolates, flavonoids and phenols is advisable for treating illnesses such as cancer and cardiovascular diseases. If a clinician is aware of the patient’s PTC phenotype he/she will be able to offer alternative foods with antioxidants but having the same effect as the food above nutrients. The ability to taste PTC is also useful in the protection from consumption of poisonous compounds. Persons that are homozygotes tasters or heterozygote mild tasters have an in-built mechanism that can protect them from consuming bitter and poisonous compounds that could easily be ingested by non-tasters. On the downside, persons that have a high sensitivity to the bitter compounds may opt to avoid anti-oxidant rich foods and instead consume fatty foods that would inadvertently have a negative outcome on their health. This could lead to health complications such as obesity, cancer and heart complications. Children that are sensitive tasters can also avoid eating healthy vegetables that will in turn affect their in-take of a balanced diet. concerning PTC non-tasters, it is possible that they may suffer from goiter in rates higher than the tasters; this is because the low or no sensitivity to bitter tastes ends to have the non-tasters consume very little amounts of salt hence low amounts of iodine that leads to development of goiter. (Wooding, 2006). Iodine is also important for the brain cell formation and hence children should consume sufficient amounts of iodine whether they are tasters or non-tasters. A study carried by Moberg et al. showed that is a significant relationship between the ability to taster bitter compounds and certain neurological conditions. The study showed that nontasters were more prone to developing neurological conditions such as depression, schizophrenia amongst other depressive conditions not commonly experienced by tasters. (Moberg,2007). In sum, the mutation of the PTC gene at the restriction enzyme is what differentiates populations as being tasters or non-tasters. Gel electrophoresis can confirm one’s genotype and the information used in making informed decisions in healthcare, as well as dietary planning. The natural ability to detect bitter taste is a protection mechanism for poisonous compounds though it can have negative effects in instances where non-tasters opt for low or no iodine foods. Children that are sensitive tasters should be encouraged to consume alternative foods that offer similar nutrients to the found in the bitter foods and fruits. In conclusion, the experiment was largely successful with the results obtained reflecting the similarity of student phenotype and genotypes to those of the documented European cohort. References Bufe, B., Breslin, P.S., Kuhn, C., Reed, D.R., Tharp, C.D., Slack, J.P., Kim, U., Drayna, D., Meyerhof, W., 2005. 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