Andrei Vasiliev 01040632
PTC Genotype Determination basing on DNA Samples (Obtained From Individuals with known PTC Test result) that are incubated with Restriction Enzyme (Hae III)
The ability to taste the bitter compound phenylthiocarbamide (PTC) and related chemicals is bimodal, and all human populations tested to date contain some people who can and some people who cannot taste PTC. Why this trait has been maintained in the population is uncertain but this polymorphism may influence food selection, nutritional status or thyroid metabolism. The gene product that gives rise to this phenotype is unknown, and its characterization would provide insight into the mechanism of ...view middle of the document...
The ability to taste PTC is a dominant genetic trait. The test to determine PTC sensitivity is one of the most common genetic tests on humans. There are three SNP's | (single nucleotide polymorphisms) along the gene that may render its proteins unresponsive. There is conflicting evidence as to whether this trait is a result of either dominance or incomplete dominance. Any person with a single functional copy of this gene can make the protein and is sensitive to PTC. Some studies have shown that homozygous tasters experience a more intense bitterness than people who are heterozygous; other studies have indicated that another gene may determine taste sensitivity. [1. Forrai G, Bánkövi G (1984). "Taste perception for phenylthiocarbamide and food choice—a Hungarian twin study". Acta Physiol Hung 64 (1): 33–40 ].Being aware of the PTC gene possition it is not a easy task to isolate the gene in laboratory conditions, making the methode of experiment sophisticated. Here is some information that can be usefull in the laboratory concerning |
materials, equipment and some procedural alternatives. To begin with a brief description of the initial cell lyses it is important to mention that Complete disruption of cells and release of contents from cellular debris is one of the crucial steps in the process. There is a number of different techniques used for physical disruption of cell, like: Lysis method | Description | Apparatus |
Mechanical | Waring blenderPolytron | Turning blades grind and disperse cells and tissues |
Liquid homogenization | Potter– ElvehjemHomogenizerDounce homogenizerFrench Press | Cell or tissue suspensions are sheared by forcing them through a narrow space |
Sonication | Sonicator | High frequency sound waves shear cells |
Manual grinding | Mortar and pestle | Grinding plant tissue, frozen in liquid nitrogen |
Freeze/ thaw | Freezer or dry ice/ ethanol | Repeated cycles of freezing and thawing disrupt cells through ice crystal formation |
[ 2.Demchick PH and Koch AL (1983). "The permeability of the wall fabric of Escherichia coli and Bacillus subtilis". Appl Environ Microbiol46 (4): 941–3]Another important step in the isolation and detection of PTC gene is amplification of genetic material by means of PCR. PCR is a technique to amplifya single or few copies of a piece of DNA across several orders of The Method: A: DNA purificationIn the very beginning Buccal cells were collected by scraping the inside of the mouth 10 times with a Buccal Collecting Brush. Next, 300 µl of Cell Lysis Solution were dispensed into 1.5 ml microcentrifuge tube. At the same time collection brush was removed from the handle using scissors and placed into the same tube. When last step was completed the tube was incubated at 65 C for at least 15 min (up to 60 min for maximum yield). After incubation collection brush head was removed from the Cell Lysis Solution, scraping it on the side of the tube to recover as much liquid...