Sociological Experiment
Methods
DNA Purification
DNA purification allowed us to gather pure DNA from cultured cells
in order to perform PCR. Wild type S9 human epithelial cell samples
were provided by Douglas Luckie and his Michigan State University
lab. The process to purify the DNA was followed from Qiagen’s
Generation Capture Column
Handbook based on the "Generation Capture Column Kit". The
protocol we followed was in the section specific to DNA Purification
from Cultured Cells and Cell Suspensions. Captured cells were kept
on ice until used. The frozen culture was allowed to thaw under a
warm water bath at 37 degrees Celsius. 200uL of the DNA sample
containing 5-7 x 10^6 cells/ml was added to a capture column and
incubated for 1 hour. 400uL of DNA Purification Solution 1 was added
to the sample and incubated for one minute at room temperature. This
solution was then placed in the centrifuge for ten seconds at
2,000-12,000 times the force of gravity (xg). This resulted in 600uL
of waste that was collected in a waste tube. It was then transferred
into a new waste collection tube and 400uL of DNA Purification
Solution 1 was added. The solution was incubated one minute at room
temperature. After the incubation, it was placed in the centrifuge
for ten seconds at 2,000-12,000 xg and acquired a waste volume of
400uL. To this, 200uL of DNA Elution Solution 2 was added followed
by ten more seconds in the centrifuge at the same speed. This
capture column was transferred to a clear DNA collection tube and
100uL of DNA Elution Solution 2 was added. This solution was then
incubated for ten minutes at 99 degrees Celsius and then centrifuged
one last time for 20 seconds at the same speed. Once the cells were
purified using DNA purification and DNA Elution Solution based on
the handbook, the strands were able to be analyzed using PCR.
Primer Design
Careful primer design was essential to proper annealing of the
primers to the desired bands of DNA. We first found the entire
sequence for the gene SOD1 and where the particular A4V mutation was
in the genome. Three primers were designed around the mutation. The
two forward primers both annealed to base pairs 142-163 on the SOD1
gene. The ALS A4V mutation forward primer, ALSF, only annealed to
the base pair DNA sequence if the mutation was present. To ensure
that the ALSF would not bind to the wild type DNA sequence, we
followed the Hidenobu Yaku method of forming primers and added an
intentional mismatched nucleotide base near the mutated nucleotide
base (5' GCG AGT TAT GGC GAC GAA
AGT
3'). The wild-type forward primer (WTF) only annealed to the
original DNA sequence that does not contain the ALS mutation because
it has only a single mismatch toward the 3' end (5' GCG AGT TAT GGC
GAC GAA AGC
3'). The final primer is a reverse primer that annealed from
752-773 base pairs and allowed for the amplified band to be 631 base
pairs long.
The equation used to calculate the annealing temperatures was Tm =
64.9°C + 41°C x (number of G's and C's in the primer - 16.4) / N
where N is the length of the primer (Rychlik and Rhoads, 1989).
Using the oligonucleotide annealing temperature equation, the
annealing temperature for WTF was calculated to be 56.31 degree
Celsius. The annealing temperature for both the ALSF primer and the
reverse primer was 54.36 degrees Celsius. As a control for our
experiment, we also used the two primers designed to amplify the A4V
mutation created by Rosen et al. The forward primer they used was
(5' ATA AAG TAG TCG CGG AGA CGG 3') and the reverse primer was (5'
GCC TTC TGC TCG AAA TTG ATG 3'), which form a 182 bp long band and
anneal at 60 degrees Celcius (Rosen et al, 1994). All primers,
including control and experiment primers, were ordered from the
Integrated DNA Technologies (IDT) website.
Site-Directed Mutagenesis
Mutant DNA was created through
site-directed mutagenesis. The primer sequence used was (5’ AGC GAG
TTA TGG CGA CGA AGG TCG TGT 3’) for the forward primer (SDFP) and
(5’ AGC CTG GGC GAC AGA GCG AGA CTT GGT 3’) for the reverse primer
(SDRP). Both primers were obtained from IDT like the previous
primers. The primers anneal to the 141-168 base pairs for the
forward primer and the 1762 to 1789 base pairs for the reverse
primer, allowing for a 1648 band to appear from PCR. The annealing
temperature of the forward primer was 65 degrees Celsius and the
annealing temperature for the reverse primer was 68.6 degrees
Celsius, utilizing the same method of calculation as above. The
product from PCR with these primers SDFP and SDRP, contains the ALS
A4V mutation and was the mutated DNA used later in the experiment.
PCR
To use the control primers, 3uL of
purified DNA, 1uL of control forward primer, 1uL of control reverse
primer, 1uL of Taq polymerase, 2uL of 10mM DNTPs, 1uL MgCl2, 5uL of
10X PCR buffer, and 36uL of nuclease-free water were mixed and
centrifuged for a few seconds and ran in PCR at 55 degrees Celsius.
Initial denaturing occurred at 95 degrees Celsius for 5 minutes. The
cocktail underwent 30 cycles involving three steps: denaturing,
annealing, and elongation. Denaturing also occurred at 95 degrees
Celsius for 45 seconds. Annealing occurred at 55 degrees Celsius for
45 seconds. Elongation occurred at 72 degrees Celsius for 1 minute
and a final elongation occurred at 72 degrees Celsius for 7 minutes.
The SDFP and SDRP were added in place of the control primers, while
maintaining the volumes of all the materials. This mixture was run
at 50 and 58 degrees Celsius for the annealing temperature while
maintaining the remaining temperatures and durations of PCR.
Additionally the ALSF primer and reverse primers were added in the
same proportions as before with wild type DNA and ran in PCR at 50
and 58 degrees Celsius. WTF and reverse primer were ran with the
mutated DNA from site-directed mutagenesis in the same quantities as
mentioned and run in PCR at 50 and 58 degrees Celsius.
A 2% agarose gel was created
by mixing 0.8g of agarose powder, 40mL of 1X Lithium Borate (LB)
buffer, and 1µL of 10 mg/ml ethidium bromide. The PCR products were
also dyed using 1µL of 10 mg/ml bromophenol blue to make sure that
the product is moving in the gel. A 1X Kb Plus molecular ladder by
Invitrogen was used. An electric current of approximately 210V was
passed through the gel for 20-25 minutes to migrate the products and
compare the length to the ladder.
Living with ALS
Our Living with ALS experiment involved a sociological,
psychological, and physiological aspect of ALS and how it affected
the patient and people around the patient. The sociological and
psychological view on ALS was shown through a still-photo
documentary created by our team. In addition, each member of our
research group spent a week attempting to mimic the deteriorating
effects of ALS.
For the first two days, the member wore 0.45kg (1 lb) of weight on
each arm. For the next three days, the weight doubled to 1.35kg (3
lbs) on each arm. For the final two days, the weight was raised to
2.70kg (6 lbs) per arm and the member had to use a wheelchair for
the majority of the time. This closely simulated how the
degenerative process in ALS works over the course of a couple years.
At the end of every day, each member completed the Disability Index at Stanford University's Health Assessment Questionnaire (HAQ) in order to gauge the disability severity of that day. We then plotted this index score versus time to see if there is a correlation between the deteriorating muscle use and the socio-psychological state of the member. In addition to this index, the member also completed the Roland-Morris Disability Questionnaire Index (RMQ) which was also plotted versus time. Finally, the member performed a simple exercise of raising his/her arms over his/her head as many times in a row as they could in one minute at the same time every night. The recorded number of arm raises that could be performed was plotted verse time. All three methods of measurement were analyzed with t-tests and correlation coefficients.