Abstract
This experiment was performed to determine the factors that influence the tas2r38 gene has on the PTC bitter taste receptor’s genotype was determined by electrophoresis using PCR and DNA extraction. The class data C allele frequency is slightly over 50% which matches with the map which shows similar frequency’s. Which suggests that there is a correlation between the SNPs and the bitter taster ability.
Introduction
The study of the variation in the ability to taste phenylthiocarbamide(PTC) was first discovered by A.J Fox in the 1930s when he and his co-worker discovered that they tasted different things (Fox, 1932 ). This then led to the discovery that some people could taste PTC, these people are called tasters and some people could not and these were called non-tasters. This was then studied further to estimate the frequency of taster and non-taster allele across the populations all over the world (Guo & Reed, 2001 ). Also, PTC has a strong correlation with the ability to taste naturally occurring substances like toxins (HARRIS & KALMUS, 1949 ) (Wooding, et al., 2004).
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The full name for the gene TAS2R38 is taste 2 receptors number 38 and is located at 7q34 complement and is 1,143bp long and with only one exon and it controls the ability to taste glucosinolates, a family of bitter-tasting compounds found in plants of the Brassica and this is controlled by a protein which is found in the tongue and is a seven-transmembrane g protein-coupled receptor. ( National Center for Biotechnology Information, 2018).
These three SNP make up the haplotypes PAV which is used to refer to tasters and AVI which is used to refer to non-tasters. The aim of the study is to find out what my genotype is at position 145 of TAS2R38 and find out about my bitter-taste ability’s and what my phenotype is. Another aim of the study is to test whether the class data will help support the correlation between the bitter taste ability and the SNP.
Materials and Methods
DNA extraction
The subjects were UK university students, because they were over the age of 18 and alive consent had to be given for the DNA to be extracted and analysed. This is in accordance with the human tissue act 2004(section 45 schedule 4) which states that consent must be obtained unless for cases or the prevention of crime. Consent was insured by making the subjects sign a consent form and privacy was ensured by the students all having an anonymous number.
The subject’s cheek was swabbed with a buccal swab 5-6 times to ensure that there was plenty of DNA cells. It was then placed in a 1.5ml Eppendorf tube which has 200ul of Phosphate Buffered Saline solution (PBS). This was then left so the PBS can lyse the extracted DNA.
20ul of proteinase K and 400μl lysis buffer (from Gene Jet Whole Blood DNA Purification Kit) was pipetted into the PBS and was incubated at 56oc for 10 minutes because this is the optimum temperature for the proteinase K to start to digestive native proteins which happened between 50-60 oc (Farrell Jr. , 2010).
The Buccal tip is then squeezed to get as much liquid out as possible then disposed of.200ul of ethanol is then pipetted into the tube and inverted, this is to ensure all the liquids are mixed properly. The Gene JET Genomic DNA Purification Columns are then secured to a collection tube. Then all the liquid from the Eppendorf tube is pipetted into the purification column and is placed in the centrifuge for 1min at 8000rmp. This is to help purify the DNA because it will bind to the collection tube while the waste goes into the collection tube.
The flow-through is disposed of and the collection tube secured,500ul of wash buffer I(from Gene Jet Whole Blood DNA Purification Kit ) was pipetted into the spin column and centrifuged for 1 min at 10,000 rpm. When finished the flow through is disposed of,500ul wash buffer II (from Gene Jet Whole Blood DNA Purification Kit )was pipetted into the column and centrifuged for 3mins at the max speed (≥13,000 rpm)
The purification tube is then filled with 50ul of elution buffet I(from Gene Jet Whole Blood DNA Purification Kit ) in the centre of the membrane to eluate genomic DNA. This is then left at room temp for 2 mins so the DNA can dissolve the buffer. Then it is centrifuged for 1min at 10,000rmp.
DNA amplification
Two PCR tubes are handed out to each subject; one is for the PCR and one is the negative control. 12.5μl of PCR master mix(made by Promega) is added to each. The PCR master mix is made up of Taq Polymerase, dNTPs, MgCl2 and reaction buffers. Then 2.5μl of distilled water,2.5μl forward primer (PTC145-F) into each tube and 2.5μl reverse primer (PTC145-R) were pipetted into each tube.
Then 5μl of the subject extracted DNA was pipetted into tube 1 and 5μl of distilled water is added to the negative control into tube 2.these are then both vortexed to ensure all is mixed well.
They are then placed in the PCR machine where it runs cycles of 95°C for 3 mins then 35 cycles of (95°C 30s, 58°C 45s, 72°C 45s), 72°C 5 min.
This is the DNA amplified.
Digestion
Digestion of 500 ng PCR product with 10 U of HaeIII enzyme by NEB (excess enzyme to make it cuts) at 37°C for 2 hours on a thermocycler and inactivated at 80°C for 30 min.
Electrophoresis and genotyping
The gel(2% agarose gel in TBE with GelRed solution) had to be checked and the comb removed.2ul of blue loading dye was added and mixed to all the undigested and digested PCR product. Then 12 of a DNA size marker was added to the far left. This is to help after the electrophoresis to count the ladder position and location. Then the samples were loaded into the gel wells. The electrophoresis tank was then connected to the power supply and ran for 30 minutes at 130v. This is then taken to the gel analysis system which will adjust the focus and brightness and switch to the UV light and the image from this is then saved.
DISCUSSION
My results were inconclusive. This means that I don’t know my genotype, however, I did predict my phenotype based on my experience and on research. It was stated that tasters would find cruciferous vegetable that contains either isothiocyanates and gitorin bitter because the there structure is similar to the structure of PTC (Wooding, et al., 2004). There is also research that states that PTC tasters are less likely to smoker being (38.4%) than in non-smokers (43.1%) (Risso, et al., 2016).AVI non-tasters were reported to have higher alcohol use than either intermediate and taster (Duffy, et al., 2004 ). Based on the research I predicted I was a taster. I predicted this because I don’t smoke or drink and I think that cabbage and broccoli are slightly bitter. This is backed up by the global diversity of the TAS2R38 PAV, AVI and AAI haplotypes from populations (Risso, et al., 2016).
I had troubles that made it so that my experiment didn’t work. One trouble was that the liquid kept bubbling which means I could have not put the right amount of my DNA or other material in the tubes which means that the PCR didn’t work and that the electrophoresis couldn’t happen. Next time I will hold the pipette all the way down and make sure to not release the lever before I have to.
Another problem was that there might not have been enough DNA for the PCR or electrophoresis to work this is because I could have not scraped my cheeks with the buccal swab enough times.Next time I will make sure to swipe more times to be certain there is enough DNA on the buccal swab.
The last problem is that on the first time I could have made it so that the liquid was pipetted under the well or on top of the well in the electrophoresis meaning that it wouldn’t show as ladders clearly when it was scanned. Next time I will make sure I’m in the well and not on top of it before I pipette the DNA.
There were some ethical considerations that had to be considered before the experiment could take place.one of the ethical issues is a person’s right to privacy and this was dealt with by each student being given a random number to put on their samples and this ensured that the result was kept anonymise. Another issue was consent which had to be given because everyone was over 18,competent and alive. This issue was handled by ensuring that everyone signed a consent form.
In conclusion I believe that the data all confirm that the TAS2R38 gene does affect the ability to the bitter taste receptor and I believe that it is a useful gene as it helps to detect the bitter taste of toxins and some poisons. The class data also helped with accepting the distribution of tastes, intermediate and non-tasters across the globe.
References
- National Center for Biotechnology Information, 2018. TAS2R38 taste 2 receptor member 38 [ Homo sapiens (human) ]. [Online] Available at: https://www.ncbi.nlm.nih.gov/[Accessed 30 12 2018].
- Duffy, V. B. et al., 2004 . Bitter Receptor Gene (TAS2R38), 6-n-Propylthiouracil (PROP) Bitterness and Alcohol Intake. Alcoholism: Clinical and Experimental Research, 28(11), p. 1629–1637.
- Farrell Jr. , R. E., 2010. Resilient Ribonucleases. In: RNA Methodologies: Laboratory Guide for Isolation and Characterization . s.l.:Academic Press, pp. 155-172.
- Fox, A. L., 1932 . The Relationship between Chemical Constitution and Taste. PNAS, 18(1), p. 115–120.
- Guo, S.-W. & Reed, D. R., 2001 . The genetics of phenylthiocarbamide perception. Annals of Human Biology, 28(2), p. 111–142.
- HARRIS, H. & KALMUS, H., 1949 . Chemical specificity in genetical differences of taste sensitivity.. Annals Of Eugenics, 15(1), pp. 32-45.
- Risso, D. S. et al., 2016. Genetic Variation in the TAS2R38 Bitter Taste Receptor and Smoking Behaviors. PLOS One, 11(10).
- Risso, D. S. et al., 2016. Global diversity in the TAS2R38 bitter taste receptor: revisiting a classic evolutionary PROPosal. Scientific Reports, 6(1).
- Rodriguez, , S., Gaunt , T. R. & Day, I. N. M., n.d.
- Rodriguez, S., Gaunt , T. R. & Day, I. N. M., 2009. Hardy-Weinberg Equilibrium Testing of Biological Ascertainment for Mendelian Randomization Studies. American Journal of Epidemiology, 169(4), pp. 505-514.
- Wooding, S. et al., 2004. Natural Selection and Molecular Evolution in PTC, a Bitter-Taste Receptor Gene. American Journal of Human Genetics, 74(4), p. 637–646.
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