Taste perception heavily influence in determining individual’s food preference, the ability to perceive tastes is linked to genetic, these include umami, sweet, bitter, sour or salty so understanding the genetic maybe the key to prevent risk of chronic disease. On the surface of the tongue there is various taste receptors such as T1R receptors linked to sweet and umami taste perception, T2R receptor which is linked to bitter taste perception, PKD1L3 and PKD2L1 ion channel linked to sour taste perception and TRPV1 sodium channel which is linked to salty taste perception.
SNPs (Single Nucleotide Polymorphisms) is a DNA sequence where variation of nucleotide differs. For bitter taste receptors in mammals there are approximately 30 genes, TAS2R38 gene codes the perception of bitter taste of individuals along with PTC (Phenylthiocarbamide) taste receptor. These combined gives these possible outcome haplotypes, homozygotic non-taster (tt) and homozygotic taster (TT) but also heterozygotic (Tt). In this practical two tests was done, gel electrophoresis and using PTC paper strip to conclude the individuals taste perception.
Saline solution (0.9%) was poured into individuals mouth and swirled for 30 seconds, it was expelled onto a universal tube. The mixture was swirled and transferred to a labelled eppendorf tube using a pipette. Using a microcentrifuge for 2 minutes at 13.5k rmp the cell pellet was separated and extracted. 100 ?l of chelex resin solution was added and placed on a hot block set to 100°C for 10 minutes. The microcentrifuge process was repeated again, 30 ?l of supernatant was removed, the solution is placed in a new labelled eppendorf tube and placed on ice.
PCR tube was labelled by individuals initial, 14.5 ?l of PCR mastermix, 5.5 ?l of water and 5 ?l of individuals DNA sample was added. Using a pipette, the solution is mixed by gently transposing the pipettes position and the solution is placed onto the thermal cycler machine at 94 °C for 5 minutes, 94 °C for 30 seconds, 64 °C for 45 seconds, 72 °C for 45 seconds, 72 °C for 5 minutes and 4 °C hold for 35 cycles.
The individual’s PCR tube is collected and placed onto the ice, 10 ?l of the PCR solution was transferred to a fresh eppendorf tube and labelled it with the individual initial and undigested. The remaining 15 ?l of PCR solution is transferred onto a fresh eppendorf tube and labelled it with the individual initial and digested, the demonstrator added restriction enzyme Hae III as it cleaves the DNA at the position where the GGCC sequence is found. The eppendord digested tube is placed into the hot block to digest at 37 °C for 20 minutes and after placed onto ice.
Gel electrophoresis is used to separate the DNA as it is negatively charged thus applying an electric field will allow DNA to move through the gel, consequently smaller DNA fragment moves further whereas larger DNA fragment travels less. 2 ?l of 6X loading dye is added to eppendorf undigested tube and mixed by transposing the pipette position, whereas for the eppendorf digested tube, 4 ?l of 6X loading dye is added and mixed using the similar process. 10 ?l of DNA ladder is loaded onto the gel, the undigested and digested samples are loaded onto fresh wells. Electrodes are placed on each end, cathode being near the wells and anode being further away from the sample, 130 V potential is applied for approximately for 40 minutes. The sample is later taken by the demonstrator and picture provided which is taken under an ultra violet light this is because Ethidium bromide is added due its ability to bind to DNA and making the fragment illuminates under ultra violet light
To compare the result of the gel electrophoresis an additional test was taken using PTC paper strip, the strip is placed in the individuals tongue until it dissolves. The response of the individual was taken under three categories mildly bitter, very bitter and no taste at all.
tt non-taster (3)
TT Taster (1)
tt non-taster (2)
As seen on the Gel electrophoresis results different size fragments are displaced at different distances, there are two homozygotic non-taster (tt) and a homozygotic taster (TT). The reason there is different patterns in the result is because the Hae III identifies sequence GGCC, allowing the taster allele to be cleaved whereas the non-taster allele reads GGGC and is not cut. The PTC test resulted as following individual 1 perceived it very bitter whereas individual 2 and 3 perceived no taste at all.
The (tt) non-taster shown a single band on both digested and undigested in 211bp whereas the taster will have 2 bands in digested DNA, one at 177bp and 44bp, the 44bp maybe fainter. The heterozygous (Tt) has three bands in the digested DNA one at 211bp, 177bp and 44bp.
As seen the gel electrophoresis result is a test for genotype as the TAS2R38 gene is responsible for the bitter perception a phenotype. The individual with the genotype, homozygotic TT is likely to perceive the PTC paper as bitter then individual with genotype homozygotic tt.
The test shows that there is a strong correlation between taste perceived when tasting PTC paper and the associated genotype as individual with TT perceived it bitter and individual with tt perceived no taste at all. This has a strong implication in medicine as SNP may help predict response of certain drugs, risk of certain diseases and environmental factors. Also, the data collected has a limited sample,33% tasted bitter strongly and 66% had no perception the other result was too faint to see.