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Successful Genotypic Cloning via PCR of the SOD1 Gene Using S9 Homo Sapien Cells for ALS Research

 

 

Authors: Jennifer Shields, George Hyde, David Rauch, Zachary Sundin

 

 

 

 

 

 

 

LB 145 Cell and Organismal Biology

Professor Luckie

 

 

 

 

Abstract

Authored by: Zachary Sundin

Revised by: George Hyde

Finalized by: Jennifer Shields

 

Amyotrophic lateral sclerosis (ALS) is a degenerative disease affecting neurons of the motor system.  To understand ALS, the experimental purpose was to clone the SOD1 gene using published methods and primers, to simulate living with ALS including associated socio-psychological aspects, and to examine any similarities between ALS related genes in homologous organisms.  We hypothesized that manipulation of temperatures and concentrations in PCR would lead to successful amplification of SOD1, which would be seen at 1,200 bp.  Additionally, we hypothesized that social anxiety levels for persons with disabilities and bystanders would increase due to uncomfortable situations, such as wearing an oxygen mask.  Lastly, we hypothesized that, given similarities among genomes and primers of organisms with SOD1 genes homologous to that of humans, successful amplification could be seen between 350 and 400 bp (Bos taurus) and between 100 to 110 bp (Daphnia pulex).  In order to test these hypotheses, three experimental methods were designed.  First, PCR and gel electrophoresis were utilized to amplify and analyze the success of amplification of the SOD1 gene.  For the socio-psychological experiment, oxygen masks were worn for one month to class and around school, while measuring social anxiety levels, via the Liebowitz Social Anxiety Scale Test, of ourselves and others.  Finally, PCR was utilized to amplify homologous SOD1 genomes for comparison with the human genome and the success of results were analyzed using gel electrophoresis.  Original amplification of Lambda Rz gene was successful (Figure 1), as well as the amplification of E. coli 16S gene (Figure 2).  Amplification of the Human SOD1 gene showed a faint band at 1229 bp (Figure 5).  Genomic amplification of both Daphnia pulex and Bos taurus illustrated primer dimer—a scenario were the primers bind to themselves (Figure 6).  In addition, the psycho-sociological findings were that social anxiety levels of all members increased over the 30 days—however, the anxiety levels began to return to the baseline towards the end of experimentation (Figures 7) (Figure 8). The overall significance is to gain further research information of the wild-type genome sequence for further comparison to mutated genome sequence—by comparing PCR products.

 

 

 

Results

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Figure 1. Amplification of the Human SOD1 gene via Polymerase Chain Reaction (PCR), which was analyzed by Gel Electrophoresis. (A) A target DNA sequence of 1,227 base pairs (bp) was amplified utilizing PCR—in which the two primers hSOD1F and hSOD1R were utilized, along with purified DNA (A260/280 ratio = 1.876 and A260/280 = 1.978). In order to run PCR, cocktails were carefully made. In this case two Master Mixes were made containing 360μL of nuclease-free H2O, 50μL of 10X PCR Buffer, 10μL of MgSO4, 10μL of dNTPs, 10μL each primer (hSOD1F and hSOD1R), 30μL of purified DNA (cocktail 1 containing DNA from Trial 4 and cocktail 2 containing DNA from Trial 3), and 10μL of Taq polymerase—each ingredient added in this precise order. Each Master Mix was used to fill 3 PCR tubes with 40μL. The PCR tubes containing the PCR cocktails were ran through the Thermo-cycler at 95ˇC for 5 minutes (initial denaturing phase). Next the PCR tubes were cycled at 95ˇC for 45 seconds, followed by an annealing phased for 45 seconds, and an extension phases for 1 minute. The reaction was cycled through 30 times. A final extension phase of 72ˇC for 7 minutes was utilized. The annealing temperature was calculated at 53ˇC (as shown above the wells). Once finished, the Thermo-cycler was then set to 15ˇC until turned off. In order to analyze the PCR, a 1.0% agarose gel was made using 2.5mL of 10X TBE (Tris/Borate/EDTA) buffer, 0.4g of agarose, 42.5mL of DI H2O (from tap)—this mixture was heated in the microwave for 57 seconds. Once cooled, 1.0μL of GloGreen was added to the gel mixture, which was then poured into a mold to solidify. Once solid, 3μL of Bromophenol Blue dye and 5μL of 1Kb+ ladder were pipetted into well 1. Pipetted into wells 2 through 4 were 3μL of Bromophenol Blue dye and 7μL of finished PCR cocktail 1. Pipetted into wells 5 through 7 were 3μL Bromophenol Blue dye and 7μL of finished PCR cocktail 2.  The gel was then run through electrophoresis at 100V for 30 minutes. The gel was observed under an ultraviolet light in order to detect bands of the target DNA sequence. (B) Migration distance (cm) vs. band size of the 1Kb+ DNA ladder utilized in order to analyze PCR product from Human SOD1 gene amplification. A target sequence of the Human SOD1 gene was amplified via PCR. A 1.0% agarose gel was made, using TBE (Tris/Borate/EDTA) buffer, agarose, DI H2O, and GloGreen, to analyze the product. In well 1, 5μL of 1Kb+ DNA ladder was pipetted and analyzed via a Semi-log plot (shown above)—in which the x-axis is the distance the bands migrated from the wells (in centimeters) and the y-axis is the length of the respected band (in base pairs).  A regression line was used for further analysis of our distinct bands. The equation from the regression line (shown above) was utilized to calculate the unknown base pair length of our PCR product. The known distance traveled was inputted (x-value), which gave a corresponding base pair length (y-value). In this case, the bands in wells 2 through 8 travelled an average distance of 3.41cm—producing a product of 1227 bp. In order to test the correctness of our regression line, the R2 value was calculated to be 0.97749—an R2 value of 1.00 is a perfect fit.

 

 

 

 

30-Day Research

 

 

 

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Navigate to:     https://youtu.be/dFfLChRs1rw

 

 

 

 

 

Discussion

 

Written by: George Hyde

Revised by: Zachary Sundin

Finalized by: David Rauch

 

Experimental Summary

Amyotrophic lateral sclerosis (ALS) is a dominantly heritable, autosomal disease that causes the weakening of muscles and ultimately death (Orrell, 1999). Research has shown a correlation between ALS and a mutation in the superoxide dismutase (SOD1) gene. Mutations cause the SOD1 gene to malfunction, inflicting buildup of reactive oxygen species. The degenerative effects of ALS targets motor neurons, leading to denervation of skeletal muscle, inducing paralysis and death (Rosen et al., 1994). To further our understanding of the SOD1 gene, PCR was utilized to amplify human S9 DNA for SOD1 specifically. The purpose of this was to use published protocols to successfully amplify the SOD1 gene. We hypothesized that manipulation of temperatures and concentrations in PCR would lead to successful amplification of SOD1, which would be seen at 1,200 bp.

In addition to PCR of the human SOD1 (hSOD1), we extended our research to compare the SOD1 gene in homologous species, Daphnia pulex and Bos taurus. Both speciesŐ SOD1 genomes were compatible with the hSOD1 genomic sequence. The amplification of these genes was performed using designed primers respective to the specieŐs genome. We hypothesized PCR would be successful due to the similarities between the homologous sequences and primers (Colbourne et al., 2011).

In order to understand the psychological and sociological aspects that accompany ALS, we furthered our research to simulate living with a symptom. Four oxygen masks and two mini-oxygen tanks were acquired from Regional Medical Imaging, which simulated the respiratory dysfunction—a major cause of death among ALS patients (Yamauchi et al., 2014). The purpose was to investigate the significance between persons with disabilities, specifically those with ALS, and social anxiety. It was found that people with disabilities are more anxious than those without, which led to the experimental design of testing each group memberŐs social anxiety with and without an oxygen mask using the Liebowitz Social Anxiety Test (Heimburg, 2002). The anxiety of those around us was also tested; it was discovered that bystanders feel more anxious when around a person with disability. We hypothesized that social anxiety levels will increase for both parties (person with disability and bystander) because of the innately awkward and uncomfortable environment that arises from these situations (Cowden, 2010).

Original Predictions

Through amplification of Homo sapien S9 DNA via PCR, the length of the target DNA segment was analyzed with gel electrophoresis to identify the amplified product. Using the primers and protocol from our published source, we predicted the final product to be at 1,200 bp because of the primersŐ specificity of binding location on the gene (Prudencio et al., 2010). To observe the gene across species, we performed the hSOD1 PCR protocol on the DNA of Daphnia pulex and Bos taurus, using designed primers derived from the hSOD1 primers, which were the dSOD1F, dSOD1R, bSOD1F, and bSOD1R. We predicted that a successful amplification of Daphnia SOD1 and Bos taurus SOD1 would be 106 bp and 370 bp, respectively, because, as Homo sapiens are homologs, cocktail and procedure manipulations of the successful hSOD1 procedure would result in success (Lyu et al., 2013) (Mossallam, 2011).  Extending our research beyond laboratory studies, we examined the sociological affects of having disabilities triggered by ALS. For our 30-day experiment we predicted that the anxiety of each group member, as well as bystanders, would increase while wearing oxygen masks because of the uncomfortable situation both groups would endure (Cowden, 2010).

Results and Ultimate Findings

            To determine optimal PCR conditions for the Lambda and E. coli positive controls, extensive trials were run to determine appropriate DNA concentrations, primer concentrations, and annealing temperatures. A master mix of 10x the original cocktail concentrations was found to be the best method for creating both cocktails (Abilock and Stephenson, 2001) (Sabat et al., 2000). The master mix contained MgSO4, which was not previously used in other cocktails; the MgSO4 helped the Taq polymerase functional efficiently as it is a coenzyme particular to Taq. The cocktails were run with two separate gradients (refer to Methods), which provided a better range for annealing temperatures to ensure successful annealing. The resulting gel for E. coli had bands at 497 bp. Using the migration distance of known band lengths via a semi-log plot, the amplified product band size was determined and supported these findings. The successful amplification of the 1Rz1 gene in the Lambda gel was shown by bright bands at 482 bp. Similar to the E. coli experiment, a semi-log plot was utilized to provide evidence of the amplified product length.

            To obtain the most pure DNA, purification required multiple trials to have an A260/280 value in the range of 1.8nm to 2.0nm. The A260/280 value represents the amount of DNA present compared to the amount of protein present within the cellular solution based on the absorbance spectrum, 260nm indicating DNA and 280nm indicating protein. For trials 1 and 2, the A260/280 values were above 2.0nm, whereas trials 3 and 4 possessed values below 2.0nm. The only trial utilized was trial 4 with an A260/280 value of 1.876nm and a DNA concentration of 179mg/ mL. In addition to the S9 Homo sapien DNA, Daphnia pulex DNA and Bos taurus DNA were purified in one attempt, with A260/280 values below 1.9nm. The DNA concentration for Daphnia was lower than expected (44mg/ mL) due to performing colony PCR of the Daphnia as opposed to DNA purification of Daphnia cells. In contrast, the concentration for Bos taurus was 94mg/ mL.

            With limited resources of purified DNA, fewer trials were run with the human cocktail. Three trials contained different annealing temperatures and concentrations. In the third trial, run at 53oC, the gel showed faint bands. A migration semi-log plot was used to support the bands and provide an accurate product length at 1227 bp. Smearing is present in the gel, signifying there was non-specific binding that could be eliminated with more accurate annealing temperatures. Successful amplification of human SOD1 gene was a control for the amplification of Daphnia and Bos taurus SOD1 genes. Three trials were performed with the homolog DNA.  No successful bands were present for either homolog. The gel showed DNA bands of Bos taurus at 15,509 bp, indicating non-specific binding, and primer dimer bands at 82 bp in Daphnia wells. A migration semi-log plot was used to find the base pair length in the final gel. Future trials would be needed to find the correct annealing temperatures and proper DNA concentrations to ensure successful amplification. To experience life with ALS, a 30-day experiment was conducted to test correlation between anxiety and disability. Group members wore the mask consistently in multiple environments to obtain the largest amount of encompassing data possible. The anxiety of each group member and bystanders was measured with the Liebowitz Test. The anxiety of group members was averaged over the entire 30 days and on a weekly basis. The data showed that overall anxiety levels elevated while wearing the oxygen mask due to the uncomfortability of an unusual situation. However, when analyzing the anxiety levels on a weekly basis the data showed that anxiety levels began to return to baseline levels as time progressed because we grew accustomed living with the disease.