This method can help answer key questions in the thrombosis and hemostasis field identifying subjects more susceptible to atherothrombotic processes in particular high-risk patients treated with aspirin. The main advantage of this technique is that it is inexpensive, simple to use, convenient, has a high efficiency and allows rapid identification of the C924T polymorphisms. Though this method can provide insight into atherothrombosis field, it can also be applied to other systems such as polymorphisms involving personalized medicine or specific drugs in order to understand the pharmacological individual response.
Demonstrating the procedure will be Doctor Sebastiano Ursi, a technician from our laboratory. To prepare the Tris EDTA buffer at pH 8.0, add 200 microliters of 0.5 molar EDTA and one milliliter of one molar Tris chloride in a beaker, then adjust the volume to 100 milliliters with sterile water and store at room temperature. Next, prepare one liter of 10X stock solution of electrophoresis buffer by adding all the reagents, then store the buffer at room temperature.
Next, prepare the gel loading dye by adding all the reagents then bring the volume to 100 milliliters with sterile water. Once prepared, store the dye at four degrees Celsius for a few months. Add 450 milliliters of water to 50 milliliters of 10X TBE buffer to prepare a solution of 1X TBE buffer.
Add four grams of agarose in 200 milliliters of 1X TBE buffer together in a 600 milliliter beaker to prepare 200 milliliters of 2%agarose gel. Solidified agarose gel stored at room temperature can be redissolved over a boiling water bath at 60 degree for 15 to 20 minutes or in a microwave oven for 35 minutes by adding a small volume of water to compensate the increase in agarose concentration. Keep stirring the beaker on a magnetic mixer for approximately five minutes till the agarose is suspended then heat the agarose on a hot plate for about 10 minutes or in a microwave oven for three minutes for it to completely dissolve.
After purification of DNA, gently mix the fresh DNA samples by pipetting tubes several times. It is mandatory to obtain 10 to 50 nanograms of a good quality template DNA extracted from human samples. For this reason, we prefer to use an automated DNA purification rather than a semi-automated or manual one.
To begin purification, first quantify and calculate the purity of the DNA by measuring the absorbents at 260, 280 and 320 nanometers, then dilute the samples using sterile water. Next, calibrate the spectrophotometer. To constitute the PCR reaction, add all the reagents to prepare 25 microliters of PCR master mix in a 0.2 milliliter microamplification tube.
Then place the PCR tubes in an automated thermocycler and amplify the purified DNA samples. Once the program finishes, stop the reaction by leaving the tubes at four degrees Celsius. Add 10X enzyme buffer five units per microliter restriction enzyme in DNA's free water to a volume of 22.5 microliters.
Next, add 2.5 microliters of PCR product to a new PCR tube then add the 22.5 microliters of restriction digestion mix to the PCR product. Incubate the PCR product restriction digestion master mix at 37 degrees Celsius for four hours, then add 0.5 microgram per milliliter of Ethidium bromide to stain the gel for 10 minutes. Pour the gel on the gel tray with the well comb in place for the gel to solidify.
Now pour the gel box with 1X TBE buffer till the gel is covered. Use fine tips to load six microliters of the amplified digested DNA in the wells formed in the gel, then add a DNA marker too in a separate well parallel with the sample. Next, switch on the electrophoresis unit.
In order to identify the C924T genetic polymorphism in the TBXA2R gene, RFLP analysis is done. In the gel, it displays two bands at 395 and 144 base pairs for the wild-type version of the major allele, whereas a single band at 539 base pairs for the mutant version of the minor allele is seen indicating no cut by the restriction enzyme. Interestingly, three bands at 395, 144, and 539 base pairs are present for the heterozygous allele.
Next, the C924T polymorphism is confirmed using sequence analysis. The highlighted sequence represents the polymorphism. Once mastered, this technique can be done in about eight hours if it is performed properly.
While attempting this procedure, it's important to remember that this method can only be used for a small number of snips and the for few samples in a working session. Following this procedure, other methods like snip analysis can be performed in order to answer additional questions like the importance of other pathways involving atherothrombotic processes. After its development, this technique paved the way for researchers in the field of molecular biology to explore genetic variations outstanding in this beginning development and progression.
After watching this video, you should have a good understanding of how to evaluate the Thromboxane A2 receptor genotype with regards to the C924T polymorphism using gel electrophoresis analysis of the PCR-RFLP samples. Don't forget that working with Ethidium bromide and UV light sources can be extremely hazardous and precautions such as gloves, safety glasses, or a face mask and other protective devices should always be taken while performing this procedure.
