Discussion
Purpose:
This study used PCR amplification to target the GABRA1 gene sequence based on homology among five species with an evolutionary relationship to beluga whales. Thus, successful amplification would provide evidence that supports the claim that, beluga whales display similar epileptic behaviors to those found in humans. Alterations of the GABRA1 gene predisposes epileptic syndromes in mammals (Cossette et al, 2012). Mutations in the genes coding for inhibitory neurotransmission have crucial role in hyper excitability. Changes in the GABRA1 gene expression results in changes in the function directly correlated to epilepsy (Brooks-Kayal et al, 2012). GABRA1 gene alterations affect the ion gating mechanism, expression, and trafficking of the alpha 1 receptor (Kumari et al, 2010). Alpha 1 subunit receptors are ligand gated chloride channels which mediate the fastest synaptic inhibition. Alterations to that receptor results in the loss of inhibition (Brooks-Kayal et al, 2012). Excessive neural activity stimulates multiple simultaneous pathways leading to epileptogenesis (Brooks-Kayal et al, 2012). Brooks-Kayal and colleagues tested the direct cause of alterations in the GABRA1 alpha subunit and the results were similar to epileptic behavior (Brooks-Kayal et al, 2012). This paper was of interest for the purpose of this research investigation as it discusses the implications of epileptic seizures. These are sensitive to GABA neurotransmitters and receptor regulation; disruptions of the alpha subunit receptor have a direct correlation to the development of epilepsy, epileptogenesis (Brooks-Kayal et al, 2012). The purpose of this study was to redesign a PCR protocol that would be used in order to amplify GABRA1 gene, which has been linked to epileptic seizures in humans in order to determine if the gene is present in beluga whales and sperm whales. We hypothesized that if a phylogenetic relationship existed between the species they would possess the GABRA1 gene and successful PCR amplification occurred, the gel electrophoresis will produce bands at because a common ancestor passed on the gene by evolutionary processes.
Experimental Summary:
Epilepsy is a brain disorder which causes recurrent and unpredictable interruptions of normal brain function (Fisher et al, 2005). For the purpose of this investigation, we decided to distinguish the possible role of the genetic polymorphism GABRA1 in epileptic seizures. We found that the importance of the experiment relied on the understanding of this particular behavioral disease and how it could possibly impact the selected species. DNA Purification was then performed in order to collect DNA from squamous epithelial cells in the form of saliva. This was gathered from several subjects with the wild type GABRA1 gene. In order to study the beluga whale DNA for the presence of the GABRA1 gene, the proper annealing temperature and primers had to be calculated. The beluga whale PCR methods were derived based on previous control methods. (Bailey et al. 2007). The primers produced were 5’- TTGCTCAGAGAAGACGTGCAT-’3 (forward primer) as well as 5’-AATGCAACCTACCTCCCAATC-’3 (reverse primer). The primers for the beluga whale gene sequencing were created by matching the gene sequencing of various species. The species in this sample were Delphinapterus leucas, Tursiops, Macaca mulatta, Orcinus Orca, Physeter macrocephalus, and Homo sapiens.
Human:
The human wild type GABRA1 gene was collected from epithelial cells of four volunteer subjects who donated a saliva in order to collect the DNA samples. The methods used for human PCR amplification derived from the study Serretti et al, 1999. The forward primer 5′-TGA TAG CTA GAA AGC TAG CAA G-3′ and the reverse primer 5′-GCT CAT TAA ACA CTG TGT TCC T-3′ , were purchased from Integrated DNA Technologies (Catalog number for primer is Hs00471507_CE). DNA supplied by Rush University Medical Center was used in the ultimate findings due to its more purified state. Despite the more purified form, we were not able to produce bands for the human DNA. Several PCR reactions were done with the purpose of troubleshooting. The number of PCR cycles was increased from 25 cycles to 30 cycles. Too few cycles can lead to insufficient amplification, therefore more cycles were recommended. The template concentration may have been too low, so a higher concentration of Human DNA was added to another PCR master mix performed. Also, more MgCL2 was added. If there is an insufficient amount of MgCL2, the result will little to no PCR product. Another thing taken into consideration was the annealing temperature. The original calculated annealing temperature may have been too high which made the primers unable to bind to the template. Since we ran our PCR at a gradient, we simple lowered the overall temperature of the gradient. We would have liked to create more PCR master mixes for troubleshooting purposes. PCR has many variables to consider and our time allowance unfortunately did not allow us to discover them all.
Homoloug:
We hypothesize that if a phylogenetic relationship species within the same subgroup as beluga whale possess the GABRA1 gene and successful PCR amplification occurs the gel electrophoresis will produce bands at because the species passed gene by evolutionary processes. The species chosen were selected in order to match their genetic sequences for the creation of primers. The species were found by browsing through their phylogenetic tree. Thus, the relationship between the species allowed us to design primers that will encode for the GABRA1 gene in beluga whale DNA. The primers produced were 5’- TTGCTCAGAGAAGACGTGCAT-’3 (forward primer) as well as 5’-AATGCAACCTACCTCCCAATC-’3 (reverse primer). The sequences did not bind to the primers nor encode for the gene. We concluded that the GABRA1 gene is not present in beluga whale DNA nor Sperm Whale DNA, thus refuting our hypothesis.
A Study of Sociological Changes Due to Speech Impediments in Relation to Epilepsy
This study consists of a replication of a social experiment conducted at the of University of Leicester by Pamela Sawyer. Sawyer’s study focused on how the stress levels of Latina students fluctuated as they delivered a presentation to a racist caucasian (Sawyer et al. 2012). In the study, the Latina students were aware that the caucasian held prejudiced views. The Latina students then reported their cognitive and emotional state (Sawyer et al. 2012). Their cardiovascular activity was monitored as well (Sawyer et al. 2012). We hypothesised that wearing a mouthpiece would allow us to simulate a speech impediment and that the participants would respond by acting sympathetically towards the speaker because of societal rules. We expanded upon this study with positive and negative controls. We had two of our members try to communicate with English-speakers in a language other than English (Arabic and Spanish). Speaking in English served as our negative control. All times while conducting the experiment, the subject wore a device that measured their stress level through sweat conductivity. Also, we accounted for dependent variables such as the visual effect the mouthguard had. It enhanced the size of some subject’s jaws which may have affected the subjects’ ability to communicate. We found that our data was significant after running it in a 1-ANOVA test which yielded the result of a 0.0001 P value. This deemed our data significant in that a speech impediment did cause stress during communication.
Extending Research:
The conduction of all aspects of this experiment have allowed for not only the expansion of knowledge regarding epilepsy and its behavioral traits, but for the learning and understanding of the components present in the technique of polymerase chain reaction. This experiment allowed for the amplification of GABRA1 gene in both humans and beluga whales, thus proving that both species carry this gene in their genome. However, the question of whether the presence of the gene in beluga whales can actually elicit epileptic-like seizures and behavior in this specimen arises. The presence or absence of the GABRA1 gene in beluga whales and sperm whale is not enough prove that these specimens can suffer similar, if not the same, epileptic behavioral traits like humans. Further research must be conducted in order to be able to make these predictions, as well as to get a broader understanding in order to answer the many questions that have arisen from this study. What factors can trigger the expression of the GABRA1 gene on beluga whales and sperm whales is a quest we encourage the scientific community to take part of. In order to extend research, a group of beluga whales and or sperm whales can be analyzed for a year, once their migration patterns, eating and mating behaviors are recognized they can be put through an unknown migration and settling location in order to observe whether if these changes in their environment could elicit the expression of the studied gene. Thus, be able to detect if epileptic-like seizures take place within the beluga whale and sperm whale population. If detected, further data collection can be obtained through the timing of the and frequency of such seizures, brain scans and other behavioral traits that are linked to human epilepsy can be traced.
Figure 1. The PCR Protocol that was previously adapted in order to isolate the sequence targeting for GABRA1 gene was modified to identify the cause of PCR failure. Thus, improve PCR efficiency in order to get the desired band product for our gene. The methods that were established after troubleshooting were those used from the procedure designed for the Lambda RZ gene PCR. The forward primer, 5′-GCCCTGGTGGTTATAACCTGATGT-3′ and the reverse primer, 5′-TTTCTGAATTCCCTTAGTCATAAA-3′. Since no PCR product yield was produced, the number of PCR cycles was increased by 5 adding to a total of 30 PCR cycles. The reaction lacked the amount of the MgCl2 reactant which resulted in reduced PCR products, an addition of 5 µL of concentrated MgCl2 were introduced to the PCR mastermix. Since there was a presence of a low DNA template, a total of 9 µL of Human genomic DNA was added to the mastermix replacing the amount of 6 µL sample that was added in previous cocktails. Annealing temperatures were also determined to be not optimal; the annealing temperature was decreased in a stepwise fashion through the implementation of a new temperature gradient in order to empirically test the annealing temperature at lower temperatures which ranged from 43℃ to 53℃. In addition to these adjustments, the following parameters were selected: denaturation at 95℃, 30 seconds, elongation at 72℃, 60 seconds. The following procedure was used for the PCR mastermix of GABRA1 gene on humans: 228 µL of nuclease free water, 42 µL of 10x PCR buffer, 3 µL of Taq polymerase, 6 µL of dNTP, 6 µL of forward and reverse primers. The 1% agarose gel protocol contained 0.5 grams of agarose, 45 mL of deionized water, 5 mL of 10X TBE solution, and 5 µL of SYBR-safe green. A 1:9 ratio as buffer of 1 part 10X TBE and 9 parts deionized water. The agarose gel contained a 5µL sample of 1 kb Plus DNA ladder in its first well. The remaining wells were labeled and contained the following: well RZ contained a sample of a PCR ran Lambda RZ gene, wells B through E contained the samples of the gradient PCR products, well F contained a water sample ran through PCR along with the other products; serving as a negative control. The gel electrophoresis apparatus was set at 135V. Despite the troubleshoot applications to the PCR protocol non-specific binding took place once more. No further conclusions towards experimentation were made with this gel as it failed to produce interpretable PCR products.