Phenylthiocarbamide Lab Report

Before it undergoes PCR the DNA extraction has a few steps to be done first, which is crucial to allow for amplification to begin, it needs to undergo cell lysis and denature the proteins, which then is purified (Tracey, 2017). The extracted DNA was then put into NaOH, a strong base and heated to 95 degrees Celsius, which leads to breaking down the hydrogen bonds between the nitrogenous (Tracey, 2017). Once all the previous steps have been completed, the PCR reaction can commence after mixing with primers, Taq DNA polymerase, reaction buffers and dNTPS. The Thermocycler was utilized to facilitate PCR making it faster, more reliable and cheaper (Tracey, 2017). The sample will undergo 35 amplification cycles, producing 3.44x1010 target sequences [ 235= 3.44x1010

Figure 1 Gel Electrophoresis for Replication Taster PTC. The gel is composed of an ethidium bromide stained 3% agarose gel demonstrating DNA fragments which were a depiction of PCR amplification. The agarose gel contains nine loading samples, including from left to right, the MW marker lane 1 precision mol mass standard, lane 2 TB undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 3 TB digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 4 TB A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 5 TB A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye), lane 6 MG undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 7 MG digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 8 MG A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 9 MG A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye).

The DNA extraction results, along with the PCR product, did not fare well. There was not enough product produced to be viable in the later stages of the experiment, so a backup was used in place of the original product.

The gel results conclude that student 1, S1, student 2, S2, and student 3, S3, all have DNA that is approximately 303 base pairs long, signifying that they all possessed the homozygous allele for the uncut DNA. The uncut DNA was represented by the recessive allele, t, and therefore the genotype of the students would be tt. Phenotypically, the students would be non-tasters of the bitter phenylthiocarbamide on the PTC

Phenylthiocarbamide, sometimes also refers to as PTC, is an organic compound that permits individuals to distinguish their level of acidic taste. Not everyone can taste it, and the individuals who can are called "tasters" while the individuals who can 't are "non-tasters". Towards the start of the investigation a PTC paper of 0.003mg fixation was given to a population of 23 participants with a specific end goal to shape two unique gatherings: PTC tasters and non-tasters. This investigation required a total of two trials. In which, every participant started by rinsing their mouths with water and

In the following labs, we were planning on discovering the distribution of the tasting allele among our laboratory class using a taste test, PTC strips and running our own DNA for the specific gene. We used the restriction enzyme to cut the DNA, revealing our tasting/non-tasting genotype. Our class hypothesis was that the taste test and the PTC strips would be a good indicator of what our genotype may be. If we found certain foods bitter and identified the PTC strip from the control by taste, then we would be carrying the gene for

NaOH is then applied for cell lysis and the ‘unzipping’ of dsDNA to ssDNA. The ssDNA may then be used to isolate and replicate the PCR product through the use of PCR and site specific primers, using 2 specific primers to isolate both sides and ends of the mtDNA D.loop, multiple runs of PCR are taken to receive multiple copies of the PCR product. The following sequence primers are used to isolate the PCR

Due to the multiple stages that are characteristic of the experiments involved in getting the results above; DNA extraction, PCR reactions, restriction enzymes/thermo cycler and gel electrophoresis, problems affecting the result might arise in the experiments. Providers of the products (instrumentation and reagents) and protocols used in the experiments often provide a list of possible problems that might arise and the troubleshooting guidelines (website 4, 5). In the above experiments DNA was extracted from buccal cells in the cheeks, the issues here might arise if food particles in the cheeks contaminate the DNA; to circumvent this it might be necessary to extract DNA before any food consumption alternative blood can be used to extract DNA but this is usually too invasive for general teaching and research laboratory.

Total RNA was extracted using the Trizol extraction kit (Invitrogen, Carlsbad, CA). First-Strand Synthesis System for RT-PCR (Invitrogen) was used to synthesize cDNA from 1.5 μg total RNA according to the oligo (dT) version of the protocol. Real-time PCR was performed using CFX Fast real-time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA). The following cycle parameters were used for all experiments: 20s at 94°C, 30s at 60°C, and 30s at 72°C for a total of 45 cycles. The relative level of mRNA for a specific gene was normalized to GAPDH levels. Table 1 shows the sequences for all primer sets used in these

Experiment 6: The DNA concentration after TAS2R38 PCR purification and quantification was 0.344 ng/µL (34.4 ng/mL). My genotype at the TAS2R38 locus was not determined by the sequencing. The genotypic frequencies of the class obtained by sequencing: 0.4 (40%) TT, 0.4 (40%) Tt, 0.2 (20%) tt. The allelic frequencies of the class obtained by sequencing: 0.6 (60%) T and 0.4 (40%)

In order for these events to occur forward and reverse oligonucleotide primers, nucleotides (dNTPs), and Taq polymerase must to be added to the PCR solution. The oligonulceotide primers complimentary to the target sequence and can be used as probes to detect the target sequence of DNA that will be used in PCR (Cox et al., 2012). The dNTPs act as building blocks for the new DNA strands that will be amplified and Taq polymerase is a thermostable enzyme used to create new DNA strands from the template (Cox et al., 2012)

For -629C/A polymorphism, 5µl of PCR products were incubated with 0.4U of Van91I restriction enzyme at 37 °C for 48 h. The thermocycler conditions after optimizing the technique for -629 C/A were: initial denaturation at 96°C for 5 min followed by 35 cycles of amplification, each cycle consisting of 45 s at 96 °C, 45s at 58 °C and 45 s at 72 °C, in a PTC- 200 MJ-Research Peltier thermocycler. The thermocycler conditions after optimizing the technique for Taq1B were: initial denaturation at 96 °C for 5 min followed by 35 cycles of amplification, each cycle consisting of 45 s at 96 °C, 45s at 56 °C and 45 s at 72 °C, in a PTC- 200 MJ-Research Peltier thermocycler. The reactions for both polymorphisms ended with an additional 7 min of extension at 72° C. The Taq1b PCR resulted in an amplified product of 535 bp and was digested using the restriction enzyme Taq1B. The presence of the Taq1B restriction site produces the B1 allele pattern

Phenylthiocarbamide or PTC is a compound well known for it’s unique taste or in some cases lack of. Depending on the genetics of an individual, PTC can taste very bitter. Likewise however, if the individual is not a carrier for the gene than he/she will taste nothing. PTC’s unique ability was first discovered in 1931 by a chemist, Arthur Fox, and his partner, C. R. Noller. When PTC powder was dispersed in the air, Noller complained of a very bitter taste however Fox was unable to taste it. Initially curious, the duo tested other individuals and found that every person could be classified into two groups: tasters and non-tasters. (Wooding, 2006) Simply, If you are a homozygous dominant or a heterozygous than you are a carrier for the gene that

Phenylthiocarbamide, known as PTC, is a compound that is sensitive to the bitter taste of some individuals while tasteless or slightly less bitter to others. The ability to taste PTC is inherited by the presence of a T2R taste receptors found on the tongue. Studies on this began during the 1930s, where scientists noticed that crystals of PTC had different affects on the taste buds of individuals. To test this observation out they used PTC-saturated paper, where the experimentee would place the paper in their mouth for a few seconds and the results were recorded. Those who were able to taste the bitterness of the PTC-saturated paper are known as “tasters”, while those who were not able to taste anything are called “non-tasters”. Being able to

Each human carries two copies of the PTC gene, which whose combination justifies whether the individual can taste the bitterness or not (Drayna, 2005). There are two alleles of the PTC gene in which an individual can carry two taster alleles (TT), two non-taster alleles (tt), or one of each (Tt). The allelic variation that detects the PTC tasting is located in over 5 to 7 chromosomes (Bufe, 2005). Like many others traits, the sensitivity to PTC is hereditary (Suzuki et al., 2010). There are specific genotypes that determine the individual’s ability to taste the bitterness of the PTC. If the bitterness can be tasted, it means the individual has a dominant allele inherited from either parent (Wooding et al., 2004). The ability to taste PTC has been widely studied as a Mendelian trait. Surveys have been done regarding the genetics of taste by classifying the tasters and non-tasters of PTC. By analyzing the statistical results, it was discovered that the ability to taste PTC is dominant. However, this may vary between men and women. Studies have also shown that women are more sensitive to PTC than men, therefore, it may be that PTC tasting is related to the levels of dithiotyrosine in the saliva. If only certain people can taste the chemicals of PTC bitterly, then more women will be able to taste PTC then men