Introduction:
Phenylthiocarbamide (PTC) taste is a well-known hereditary characteristic that differs from person to person. On chromosome 7, the PTC taste receptor gene, or TAS2R38, codes for the taste receptor protein that recognizes PTC (Risso et al., 2020). (Daily Ingestion of Cranberries) Polyphenol Oral Rinse Modifies Oral Microbiome in PROP, but Not Taste Perception The genotype at this locus, which has three primary variants: homozygous dominant (AA), heterozygous (AV), and homozygous recessive (VV), affects the capacity to taste PTC. The TAS2R38 gene will be amplified in this experiment using polymerase chain reaction (PCR), and the resultant DNA fragments will be examined on an agarose gel.
Hypothesis:
We estimate that people with the homozygous dominant genotype (AA) would have a distinct banding pattern on the agarose gel than persons with the homozygous recessive genotype (VV) based on our knowledge of the PTC genotype. Due to an additional restriction site for the enzyme HaeIII in the AA genotype, we specifically anticipate a bigger DNA fragment.
Materials and Procedures:
After washing our cheek pockets with 10 ml of saline solution, we transferred 1,000 ml of the resultant cell suspension into a microcentrifuge tube that was labeled. We next used a 30 ml pipette to resuspend the cells in saline solution after centrifuging the tube for 90 seconds. The mixture was then heated at 99°C for 10 minutes using 30 ml of the cell suspension transferred to a PCR tube containing 100 ml of chiles (Tuzim & Korolczuk, 2021). We transferred 30 ml of the resultant DNA extract to a 1.5 ml tube and put it on ice after shaking and centrifuging the tube twice for 90 seconds.
Then, we used a micropipette to inject 2.5 ml of cheek cell DNA straight to a PCR tube after adding 22.5 ml of PTC mixture. Denaturing at 94°C for 30 seconds, annealing at 64°C for 45 seconds, and extending at 72°C for 45 seconds were performed on the PCR tube in a thermal cycler set to 30 cycles. The DNA that had been amplified as a consequence was chilled.
We pipetted 10 ml of the PCR product into a 1.5 tube marked with the letter “u” for undigested in order to evaluate the PCR product. The leftover PCR product was then marked with the letter “d,” and 1 ml of HaeIII was immediately added to the tube. The reagents were combined and pooled by tapping, and then the tube was put in a thermal cycler for one cycle at 37°C for 30 minutes before being put on ice until we were ready to continue.
We applied masking tape to the ends of the gel tape and inserted a well-forming comb to view the DNA fragments on the agarose gel. We filled a third of the way up a toothed comb with 29 agaroses and let the gel 20 minutes to set. In order to generate a smooth buffer surface, we added extra TBE buffer after removing the comb from the gel and before placing it in electrophoresis (Tran et al., 2021). The left lane of the gel was loaded with 20 ml of PBR322/BstNI size markers, and 10 ml and 16 ml of ‘u’ and ‘d’ were added to various wells. We used 150V to run the gel for
Discussion
Two separate bands—one for the undigested PCR product and one for the digested PCR product—were seen on the stained gel as a consequence of the analysis. About 335 base pairs were present in the undigested product, while 165 base pairs were present in the digested product (Henslee, 2020). These results are in line with what was anticipated given the layout of the experiment and the size of the PTC gene fragment that was accessible.
The findings of the stained gel show that PTC-PCR may be used to identify the genotype for PTC taste. If a person has the dominant allele for PTC taste, HaeIII will break down the PCR product into two smaller pieces. The PCR result will not be digested and will instead stay as a single, bigger fragment if a person has the recessive allele (Yousaf et al., 2022). In this experiment, the digested band stands in for the heterozygous or homozygous dominant genotype, whereas the undigested band represents the homozygous recessive genotype.
We found that the findings of the stained gel were consistent with the outcomes anticipated by the text article when we compared them to the results. This may indicate that the experiment was properly carried out and that the findings are trustworthy. In order to isolate the gene for PTC taste, amplify it using PCR, and then digest it with HaeIII to ascertain the person’s genotype, the experiment’s four procedures were essential.
Conclusion
The experimental findings support our theory, according to which PTC-PCR may be used to ascertain the genotype for PTC taste. Whether a person has the dominant or recessive allele for PTC taste may be determined by the presence of two different bands on the stained gel, one for the undigested and one for the digested PCR result.
For PTC taste, the PTC-PCR lab is a useful tool for identifying a person’s genotype. This study reveals the genetic foundation of PTC taste and may be used to identify a person’s genotype and phenotype. The success of this experiment demonstrates the value of using molecular biology methods to isolate and examine certain genes of interest.
References
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