For a long time, the medial scientists have tussled with the baffling incidence of many people who feel phantom limb pain in post-amputation with others not experiencing such pain. Although they have unearthed that phantom limb pain, severity varies from mild to extreme pain. In this far, the causes of Phantom limb pain are undefined yet, though there are possible factors that cause the pain in the affected area. Particularly, despite the absence of the limb, the nerve endings at the site maintain their role of transmitting pain signals to the brain. Consequently, this makes brain retain a sensory map interpreted that the limb is present causing phantom limb pain. Occasionally, the brain memory retains the signal and interprets it as pain, regardless of the nature of injured nerves with varying symptoms for different people. Some people feel it as shooting, stabbing, tingling, cramping, heat, and cold in the position of amputation (Cone, 2008). Given that the symptoms are all different owing to varying causes, no success of exhaustive treatment has been reported across the entire healthcare sector. Particularly, although more than 70 methods of treating phantom limb pain have been identified; successful treatment of persistent types of pain is not commonly reported (DeLisa, Gans, & Walsh, 2005). Our lab group hypothesized that genetic material can be different between people who feel phantom limb pain and people who do not experience phantom limb pain. Therefore, this project is meant to extract DNAs from saliva samples and examining the genotypes from each of the groups identified above and find out the distinguishing factor causing phantom limb pain.
Firstly, we obtained 1000 saliva samples from people who never felt phantom limb pain, and another 1000 saliva samples from people who experienced the pain. Concurrently, the samples are mixed with the DNA Genotek kit by inverting and shaking for a few seconds gently, to ensure the materials in the samples are well distributed. Then, the samples are incubated at 50 Celsius degree in a water incubator for at least one hour and thereafter are incubated overnight. This incubating step is vital because DNAs is adequately released and the nuclei are inactivated. Consequently, incubation of samples before conducting a liquating is necessary to ensure the samples retain homogeneity.
Succeeding the incubation is a process where 500microL of the mixed sample is transferred to a 1.5mlMicrocentrifuge tube. The remainder of the sample can be stored at room temperature. For the 500microL of the sample, add 20 microL of PT-L2P to the Microcentrifuge tube and mix by vortexing for a few seconds, which makes the sample turbid as impurities and inhibitors are precipitated. After that, the sample is incubated on ice for 10 minutes. After 10 minutes, the sample is put in a centrifuge at room temperature for 10 minutes at 13000 RPM. This separates supernatant which contains DNA and pellet which contains both proteins and impurities. Now, the clear supernatant is transferred with a pipette tip into a fresh Microcentrifuge tube while the pellet containing impurities is discarded.
Next, 600 microL of 95% ethanol is added to 500 microns of supernatant and is mixed by inversion by 10 or more times at room temperature. During the mixing process with ethanol, the DNA hairy precipitation will seemingly appear depending on the amount of DNA in the sample. Following that, the sample is kept at room temperature for 10 minutes to allow the DNA fully precipitate. The sample tube is put in the micro centrifuge in a known orientation and subsequently centrifuged at room temperature for 4 minutes at 13000 rpm. At this stage, the supernatant containing ethanol is carefully removed with a pipette tip and discarded. Again, 250 microL of 70% ethanol are added to the sample tube containing pellets, and left uninterrupted for a minuteas a wash to remove residual inhibitors. After a minute, the ethanol is completely removed without disturbing the pellet. Successively, 100 microL of TE solution which is DNA storage buffer is added to the sample tube with DNA pellet and vortex for a few seconds until the pellet is dissolved in TE solution, before complete hydration of the DNA is ensured by incubating at room temperature.
Firstly, an analytic procedure using the fluorescent dyes is preferred to yield specific results compared to absorbency method which would demand initial purification of the samples with RNase to avoid potential contamination. This is essential since measuring concentration of DNA in complex mixtures is quite difficult but at 260 nm there would be maximum absorption by DNA, RNA and protein alike. Florescence method suits this analysis to generate specific quantifying of the double-stranded DNA (dsDNA) in the samples. Consequently, fluorescent dyes including PicoGreen and SYBR Green I dye as described in PD- PR- 075,alongside a micro-plate reader are suitable to ease the quantification and avoid potential interfering by RNA. Essentially, the droplet-based experimentation ensures there are no contact between the contents of the droplets and the channel walls absorption and loss of reagents on the channel walls is prevented (Solvas, Leeper, Cho, Chang, & Edel, 2011). This would also save on the cost during quantification. Instead, other kits available may also be applied such as the Invitrogen’s Quant-iT PicoGreen dsDNA. In spite of the protocol adopted, the DNA is diluted after purification with the TE solution in a portion of 1:50 while a 5 microl is used during the quantification investigative procedure.
After the extraction of DNA from saliva samples, using purification and quantification of DNA by fluorescence method, their limb related gene sequences and genotypes were compared. Here, incidence of gene mutation occurring on sequences from people who felt pain at common region from every sample DNAs is compared with sample DNAs from people who never felt phantom limb pain. This would lead to a definite conclusion that certain genetic mutation causes the phantom limb pain. Then again, When there is a specific probe that can bind to specific DNA strand sequence, it can be distinguished if the pain is caused by mutation sequences or not. For example, a specific sequence probe assumes a specific genetic mutation. In a situation where the DNA strand is denatured, the probe is bound to the paired bases of mutated DNA strands from people experiencing phantom limb pain. This probe will however not be bound to the sequence from people not experiencing the phantom limb pain.
Phantom pain is a sharp feeling which seemingly arises from the body part which in actual sense no longer exists. Phantom limb pain exists as a syndrome in perception of sensations, usually including pain, in a limb that has been amputated and a condition is experienced as if it were still attached, as the brain continues to receive messages from nerves that originally carried impulses from the missing limb (Scheinberg & Lukas, 2012). For too long, the medical community had the perception that this post-amputation phenomenon entails a psychological problem, but overtime the perception has faded with experts’ research revealing that such feelings emerge from the brain and spinal cord. In particular, the new theory outlines that causes of ghost pain stems from lasting representations of the amputated limb in the brain and linked to the disrupted activity between different parts of the sensorimotor cortex, ─ a part in the brain processing touch and movement (Lewis, 2013). Many a times, phantom pain arises in individuals with amputations, but may also arise amongst after surgeries in other body organs and parts such as breasts, tongue and eyes.
Naturally, individuals exhibit varying reactions to medications and for some instances, response to phantom pain demonstrate this fact. For example, after amputations some individuals feel better and over time the feeling subsidizes even without treatment. However, for the majority managing the phantom involves continual administration of regional analgesia and NMDA receptors, ketamine and memantime though arriving at conclusive results is an unfinished journey (Jensen, Watson, & Haythornthwaite, 2008).
Ordinarily, since the exhibiting symptoms are all unique and distinct owing to their varying causes, it has never been a simple exercise to treat phantom limb pains. Typically, phantom limb pain appearing to emerge from where an amputated limb used to be- is often excruciating and almost impossible to improve through neither medical treatments nor the destructive surgical procedures (Cole, 2004).Consequently, our lab group postulated that genetic material differs with different individuals suffering phantom-limb pain and there are individuals who will never feel such pain. For that reason, this project prioritizes extracting DNAs from different saliva samples to test the above mentioned hypothetical position. Ideally, the major obligation involves observing the genotypes extracted from the two groups including saliva from individuals encountering such pain and the rest from a group of individuals who never felt phantom limb pain. At this level, emphasizing on using the two samples enhances the process of finding out the difference between the two groups and what causes the phantom limb pain. As a result, the saliva sample from individuals not suffering from the phantom pain serves as a control experiment to certify and test the hypothesis.
Notably, the process of observing the genotype is critical thus demanding a great degree of caution to ensure the initial results are attained. For instance, among the 2000 samples of saliva required in the extraction exercise, extracting DNAs in some failed. Secondly, during the DNA purification stage, where 600 microl of 95% ethanol was added to the samples and mixed thoroughly by inverting and placing the tubes in the centrifuge, failed to generate pellets in the tube. This observation may be explained on an account of mistakes arising in the successive steps throughout the method. This affirms that the process demands a higher degree of accuracy to avoid inclusion of foreign bodies which may curtail accomplishment of the intended goal. Afterwards, the mixed sample is left incubated on ice for approximately ten minutes before successive centrifuged for another five minutes. Till when the mixed sample is left to settle after the 5 minutes of centrifugation process, that pellets and supernatant are visible. At this juncture it is essential to point out that, the supernatant should be transferred to another tube. Remarkably, when transferring the supernatant is partially done where it contains no DNAs, it is evident that there are no DNA pellets in the tube at the end of the purification stage leading to erroneous results.
- Cole, J. (2004). Phantom limb pain. Retrieved July 12, 2013, from http://www.wellcome.ac.uk/en/pain/microsite/medicine2.html
- Cone, R. E. (2008). A New Treatment for Phantom Limb Pain?: A Tri-partite Mirror Apparatus and Cognitive Behavioral Therapy. San diego: Alliant International University.
- DeLisa, J. A., Gans, B. M., & Walsh, N. E. (2005). Physical medicine and rehabilitation medicine: Principles and Practice (4 ed.). Philadelphia: Lippincott Williams & Wilkins.
- Jensen, T., Watson, P., & Haythornthwaite, J. A. (2008). Chronic Pain (Vol. 2). Broken Sound Parkway NW: CRC Press.
- Lewis, T. (2013, March 05). New Theory Explains Why Amputees Feel Phantom Pain. Retrieved July 12, 2013, from http://www.livescience.com/27641-phantom-pain-linked-to-brain-mapping.html
- Scheinberg, D., & Lukas, R. (2012, November). Phantom Limb Syndrome. Retrieved July 12, 2013, from http://www.med.nyu.edu/content?ChunkIID=96857
- Solvas, C. i., Leeper, K., Cho, S., Chang, S. I., & Edel, J. B. (2011). Fluorescence Detection Methods for Microfluidic Droplet Platforms. The Journal of Vizualized Environment.
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