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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
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
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.