The reuptake of dopamine within the brain is initiated by proteins referred to as “Dopamine Transporters” (DAT) found in-between neurons. DAT act on the pre-synaptic neurons nerve endings and allows them to absorb the dopamine neurotransmitter, thus terminating the transmission of a message. The reuptake and regulation of dopamine results in a steady and level headed mental state.
Dopamine as a molecule is a monoamine neurotransmitter, a term that refers to its chemical structure and the fact that it derives from an amino acid. Dopamine is also a Catecholamine (dopamine acts as both a neurotransmitter and a hormone) a term that also refers to its chemical structure and the fact that it contains a catechol nucleus. Dopamine synthesis occurs when the amino acid tyrosine is converted into L-Dopa which is decarboxylated to form dopamine.
There are several areas of their brain where dopamine neurons are concentrated, the largest are the substantia nigra and ventral tegmental area in the mid-brain and other areas include the hypothalamus, olfactory bulb and retina.
There are several major dopamine pathways that carry dopamine from these areas of concentration to other parts of the brain. Some of the largest are the Neostriatal/Nigrostriatal pathway, which stretches from the substantia nigra to the striatum. The mesolimbic pathway which stretches from the ventral tegmental area to the nucleus accumbens and other limbic and the structures of The Mesocortical pathway which stretches from the ventral tegmental area throughout the cerebral cortex
Abnormal dopamine levels have been associated with most commonly ADHD (1), bipolar disease (2), Parkinson’s disease (3) and various other mental conditions, and since Dopamine Transporters are the pivotal factor in regulating dopamine levels, medications act on DAT to bring dopamine levels back to normal. This is the fundamental basis on the mechanism of action of medications that aim to change dopamine levels within the brain – they’ll either suppress or stimulate these dopamine transporter proteins that aim to change dopamine levels within the brain.
To obtain DNA from buccal cells which was then used to determine the concentration and purity. Amplification of the 3’UTR DAT allowed the use of electrophoresis within an agarose gel.
Extraction of DNA from buccal cells
10ml of 0.9% saline solution in a 50ml centrifuge tube is provided, use the sterile saline solution to wash around your mouth for approximately 20 seconds (to collect buccal cells) and proceed to spit the saline solution back into the tube provided which should be labelled (name, date and solution).
Place the saline -buccal sample into a centrifuge, the important part of the centrifuge is to balance it (using similar weight samples and opposite placements within the centrifuge). Spin/centrifuge the samples at 2000 RPM for 10minutes.
Once centrifugation is done the sample will contain a pellet at the bottom of the tube and a supernatant liquid above then using a transfer pipette very carefully cyphon off the supernatant liquid as much as possible without disturbing the pellet. Remove almost all the supernatant liquid and discard the liquid into waste beakers that contain bleach (bleach pots). All that should remain is the pellet.
Using a new transfer pipette, pipette 500l of re-suspended chelex beads into your tube containing the pellet (chelex beads are an ion exchange resin which will bind to cations thus inhibiting DNases which would digest DNA).
Using the same tip re-suspend the cells by pipetting up and down several times (check that no clumps remain) and again using the same tip transfer the cell-chelex solution into a 1.5ml screw capped Eppendorf (which should be labelled with initials)
Now take the cell-chelex solution and boil for 10minutes either in a water bath or heating block (set at 95C so that the cells will lysate).Then place the tube on ice for 5 minutes to cool down.
Now the cells have become lysate, the debris and proteins must be removed by centrifugation using a micro centrifuge (once again balance the centrifuge) that span at 130000 RPM for 3 minutes to form a pellet that contains chelex and denatured proteins. The DNA is contained within the supernatant liquid therefore must be removed using a pipette into a new-clean Eppendorf tube.
Proceed to label the tube with initials then place in an ice bucket to be stored at -20C.
The results obtained from the extraction of DNA from buccal cells/DNA Quantification and purity was successful as it was within the range of 1.8 to 2.0 as the results were 1.81.
Our third practical, however, produced unattainable results as seen from figure 2 as the DNA did not project through the gel as predicted it should have, compared to the sample as seen in Fig.3. There are several reasons why the results from Figure 1 produced unattainable results.
First, the percent of the agarose within the gel (usually 0.7-3%) determines the distance between DNA bands of a length. As, a whole the lower the concentration of the agarose gel the better it is for larger molecules as lower concentration agarose gel results in greater separation between bands that are relatively close in size. However, the main disadvantage of higher concentration agarose gels is the potential of longer-run times, instead, a potential solution to high concentration agarose gels would be to either run a PFE (pulse field electrophoresis) or FIE (field inversion electrophoresis).
Second, the applied voltage, the higher the voltage, the faster the DNA molecules move. But the applied voltage has parameters by the data that the higher the voltage the more likely the gel may melt (voltage must be set between 1-10v per cm whereas the gel may melt at 5-12V per cm). During the experiment the voltage was set at 70V but yielded no results after the set time (40 minutes) whereby it was decided with the guidance of the lab technicians that a higher voltage would need to be applied, in this instance 100v was used but also yielded unattainable results as after another set time (further 40 minutes) the gel barely moved which may occur as an internal issue with the power pack given however at 120V the gel proceeded to move down and through the gel but at a potential cost as the higher voltage applied may have caused the buffer to evaporate and expose the gel (5).
Furthermore, other causes can include the fact that agarose gel’s do not have a uniform pore size, TBE (Tris-Borate-EDTA) buffer (1mm was to be set above the gel) may have been overused both in the gel and above the gel, TBE buffer is a good conductive medium so is less prone to overheating and is used for longer runs and TBE offers a high resolution and has a high buffering capacity at greater temperatures however TAE (Tris- Acetate-EDTA) buffer may be also be used as it is significantly cheaper to make and stocks can be 50x more concentrated this can result in the TAE buffer taking up less space than the original 10x concentrated TBE stock provided. Also, agarose gel electrophoresis must be run multiple times to produce clean sample results which played an important factor as running the experiment for the first time and without the capability of running it multiple times to compare different results faulted the understanding of the experimenters as they all produced inconclusive results from their first and only attempt at a clean sample.
All things considered, the amplification of the gene DAT 3’UTR VNTR by the use of the laboratory technique polymerase chain reaction (PCR) required a lot of practise and accuracy to obtain clean sample results.
Founded from the results gained from the experiment of figures 1 – 4, I believe changes will need to be made for future replication of this exact technique but it will be most important to repeat the experimentation multiple times to gain clean sample results and implement more precise methods to obtain pure DNA. One way would be to find a set way to set the concentration of the agarose gel consistently through the experiments and use (the lower the better) however if a higher concentration of agarose gel is needed polyacrylamide gel can be used at higher concentrations. Another implantation would be to set the specified voltages needed and a new power pack source if needed.
Lastly, the implantation of a new buffer could be most profound as, if a higher voltage is still required and longer runs due to better buffering capacity’s (above 80V) the TBE buffer would be the necessary buffer required however TBE buffer decreases electro phonetic mobility in agarose gel by approximately 10%(4) if necessary and dependent on circumstance and situation TAE buffer could be used for shorter runs as most agarose gels are run for a relatively short period of time and TAE buffer cane be reused for up to 4 to 5 electrophoretic separations compared to TBE buffer this intern maybe able to produce purer results