PCR
Analysis of Human IB3 Epithelial Cells to Reveal Presence of FDRA Expansion
Mutation on FXN Gene
By: Elena Vlachos
Gene Kim
Patrick Kato
Sydney Maresh
LB 145 Cell and Molecular Biology
Lab section Monday 4:10-6:10 pm
Joe Conley and Anthony Watkins
Douglas Luckie,
Ph.D.
4/22/2013
ABSTRACT
Friedreich’s ataxia (FDRA) is the
most common autosomal recessive ataxia and is primarily caused by a GAA trinucleotide
repeat on chromosome 9. To discover this mutation, it was hypothesized that it
can be revealed through PCR and electrophoresis diagnostics. The primers were
developed to anneal to the DNA with and without the presence of the expansion
because they coded for the constant sequence on either side of the expansion
location. Observing the gel Electrophoresis, the targeted area was not
amplified due to the lack of proper annealing of primers. When compared to a
known ladder, a mutant allele inspires a significantly larger than 457 bp band
because the longer chain of GAA appears higher on the ladder than the shorter
normal GAA chain (Campuzano et al
1996). The prediction theorizes that DNA sample from human epithelial IB3 cells
were homozygous wildtype (without expansion
mutation), mutant homozygous, or heterozygous (Gakh et al 2010). The first runs of the
control primers yielded smears, which could arise from degradation, low
temperatures or non-specific binding. The temperatures of the annealing phase
of PCR were increased to yield more accurate bands. In theory, there should
have been bands around 450-60 bp. Throughout
the experiment; there wasn’t any success with getting the control primers to
anneal. The magnesium concentration was altered in order to help with obtaining
bands, but that proved unsuccessful. The experiment was conducted in order to create
a diagnostic test for the medical field like that provides affected patients
with a better grasp on the severity of their disease to allow planning for future
care and precautions. To simulate the symptoms and expose the sociological
hardships related to Friedreich’s Ataxia, a 30 day experiment lived by
researchers and data was gathered. The data showed that the compounding of
symptoms had a negative difference from healthy experiences in a range
impairing quality of life by at least 50% because the normal tasks will become
impossible in some cases (Wilson et al
2007). After completing the sociological experiments the hypothesis was
confirmed participants decreased abilities to function normally decreasing
their quality of life significantly
DISCUSSION
Experiment Summary
Friedreich’s Ataxia (FA) is a
progressive degenerative disease of the peripheral nervous system caused by an
autosomal recessive mutation of the FXN gene on chromosome 9 that most commonly
results from a trinucleotide (GAA) expansion. Most common in Caucasians
(Koeppen et al 2011), this genetic
mutation creates a deficiency of the Frataxin protein that is involved with the
mitochondria. This affects the health of cellular pathways thereby altering and
slowing the cellular response and health over time and resulting in progression
of the disease (Gakh et al 2010). Wild type DNA
will only have 5 to 60 incidences of the GAA repeat, but DNA that codes for
Friedreich’s Ataxia will have anywhere from 70 to 1000 with an earlier and more
severe onset and progression of symptoms the greater the expansion (Ashizawa et al
1997).
Because of the immense variation
in number of trinucleotide repeats, a PCR assay was developed based on primers
from previously published research. These allele specific primers were used to
amplify the sample wild type DNA by annealing to the DNA sequences on either
side of the expansion area and an electrophoresis test to analyze the bands. It
was hypothesized that the primers would attach to the amplified DNA from the
human IB3 epithelial cells without the presence of the expansion because it
codes for the ever present sequence on either side, and the electrophoresis
would reveal the wild type bands for a gene without the expansion allele when
compared to a 1 kb plus ladder because the chain of GAA would appear in the
accepted region of approximately 457 base pairs (Lamont et al 1997).
Additionally, a
sociological experiment was conducted over thirty days in order to shed light
on the hardships that are a result of this genetic mutation bringing to life
the disease itself by taking on simulated expected symptoms gathered from
previous research. This was done by taking on progressively more symptoms such
as declining vision, hearing, and coordination (Campuzano et al, 1996) during the allotted time and completing simple tests.
Over the course of 30 days, two experimenters used props to simulate symptoms
while two others served as controls with no symptoms. After each symptom was
added, the list of activities that each individual could not do was recorded.
In
the first week, the experimenters simulated the effect of vision loss for at
least two hours by removing contact lenses. A Snellen chart was then used to
compare overall quality of sight between the experimenters and controls.
Following the first week, the effect of hearing loss was compounded onto the
effect of vision loss. This was done by placing cotton balls in the ears to
limit the experimenter’s hearing ability. Once again, the compounded symptoms
were in affect for at least two hours a day. At the end of the week, a decibel
test was calibrated and audible range was compared between the experimenters
and controls.
During the final two weeks, the loss of
coordination was compounded onto the preexisting conditions through improper
gait and the use of a wheelchair. The decrease in efficiency and independence
as a consequence of these symptoms was analyzed by comparing the time required
to reach a destination limping, using crutches, and using a wheelchair. The
degradation of life over a gradual time was represented in the data by the
declining number of activities the experimenters were able to do.
Predicted Results
The constructed primers and assay for the
amplification and the PCR of Human DNA will use samples of homozygous wild type.
Fprimer1 and Rprimer1 annealed to both sides of the expected area of the GAA
expansion. It was predicted that the primers would anneal to all the samples,
and the electrophoresis would show the homozygous wild type band to appear
brighter around 457 base pairs on the 1 kb plus ladder because of the shorter
normal chain of GAA trinucleotide repeats (Campuzano et al 1996), the homozygous mutant type would have clusters higher
on the ladder (around 2-5 kb) because of the longer chain (Lamont et al 1997). By examining these bands, the presence of the
mutant FXN gene would be determined.
Furthermore, the sociological experiment
was constructed and the symptoms simulated allowing prediction to be made on
the differences in quality of life experienced by patients affected by FA
versus healthy subjects. We predicted that the results of the hearing and
vision tests would show a marked difference by at least 25% worse than a
healthy subject because of the sensory nerve damage. It was also predicted that
difference in travel time would increase substantially because walking with a
disrupted gait and balance can take much longer requiring constant support and,
as a typical patient will need a wheel chair around 15 years after onset, of
symptoms, this aid can also increase time necessary to get from place to place
(Pandolfo et al
2008). Due to the progressive nature of the disease over time (Gakh et al 2010),
as more symptoms hinder the individual’s ability to perform everyday tasks,
there will be a negative impact in terms of speed or accuracy for the results
of each test.
Findings and
Implications
After conducting multiple trials of
a PCR assay along with gel electrophoresis, it was determined that successful
amplification of the desired DNA segment did not occur. The original prediction
of the primers properly annealing and amplifying was incorrect, and instead
there was an overall lack of bands on the gel electrophoresis product. When
using the selected control primers (Campuzano et al 1996), the best results were represented by a smear in the
gel electrophoresis product at an annealing temperature around 62.2°C
without altered salt concentrations. As a smear tends to indicate nonspecific
binding or degradation, it can be concluded that the annealing temperatures
used were not correct or there was degradation of the DNA. However, when
provided with a pure sample of DNA, bands appeared, but not at the desired
location. A possible explanation for nonspecific binding could be an incorrect
annealing temperature, indicating that the right temperature was never selected
and tested with. Additionally, there could have been degradation of the primers
used, which would not allow for specific binding. In terms of template
degradation, this possibility was eliminated when the group was provided with
pure DNA.
Furthermore, the observed bands
were not at the expected 457 base pair location on the 1 kb plus ladder. This
could be attributed to the lack of specificity in how many GAA repeats the DNA
used had. Due to the fact that every set of DNA has a different number of GAA
repeats, the band length for one set of DNA could be different for another.
Therefore, the bands shown in figure 4 could represent the section of amplified
DNA with very few GAA repeats. While it is unlikely that the section of DNA
would decrease from 457 to around 75 base pairs, it is still a possibility.
With future modifications and
corrections to the primers for the PCR assay, the presence of Friedreich’s
Ataxia could be determined using this test as a genetic indicator for patients
of questionable genotype. The results would follow the trend represented in
previous research on this expansion (Lamont et
al 1997) that supported studies by Campuzano that had already depicted the
mutation location and its autosomal properties. If the predictions had been
correct, the bands revealing genotype for homozygous mutant type FXN gene would
code for the expression of the disease, by the heterozygous with one affected
allele and one allele with no mutation present would not express the unhealthy
phenotype.
Apart from simply indicating the
presence of this disease, this test could also suggest the severity of the
specific patients disease by examining the band size and position of the
electrophoresis test, which would suggest the amount in of trinucleotide
repeats. As previously stated, the greater the expansion the faster the disease
will cause deterioration, and with this warning, even though there is no
current cure, the patient could obtain necessary treatment and plan for the future( Lamont et al
1997).
The 30 days experiment yielded
interesting results about the social implications of this crippling disease
using a compounding of symptoms over the course of a month.
For the first
week of the 30 days experiment, vision only was altered for the experimenters.
The experimenters’ normal, corrected vision value is 20/20 and allows them to
function normally, if not well. The subjects spent at least two hours a day for
a week with uncorrected vision, a value of 20/50, to imitate the damage to the
optical nerve that is sometimes associated with the disease. The subject could
not sit in the back of the classroom, watch movies with friends, take notes
well and put in contacts. Figure 6 demonstrates the data changes in these
instances. Following that week, vision and hearing were altered for the
experimenters. The experimenters’ normal hearing ability was impaired by
placing cotton balls in their ears. The subjects again spent at least two hours
a day for a week with impaired hearing to imitate the loss of hearing patients
experience. The subject experienced an even greater loss in the activities they
could do. Figure 6 again demonstrates
this. In the final two weeks, vision, hearing, and coordination were altered
for the experimenters. The experimenters’ normal coordination was impaired
through improper gait and the use of a wheelchair. The subjects spent around
two hours a day for two weeks with loss of coordination to imitate this
experience that patients often endure. The subject once again experienced a
great loss in the activities they could do.
Additionally,
the average time it took for the experimenters to either limp, crutch, or
wheelchair to a certain location was recorded. This time was then compared to
the times it took for the controls to walk to the same location. This data was
represented in figure 5, and it represented how patients would experience a
more difficult time getting to certain locations as their muscle conditions
deteriorated when compared to healthy individuals. To determine if the
different times were statistically significant to one another, an ANOVA test
was conducted. There were four different data sets, with them being the time it
took to get to the destination when walking, limping, crutching, and being
pushed on a wheelchair. When an ANOVA test was conducted, the p-value was
significantly below .05, which indicates that there is a significant difference
between the overall data sets. With the ANOVA test, a Tukey test was also run,
which showed that between each individual set of data, the p-value was significantly
below .05. With the overall p-value and each individual p-value below .05,
there is evidence that there is a significant difference between the times of
walking, limping, crutching, and using a wheelchair. This means that patients
who are forced to adapt to different physical ailments experience an impactful
change in how long it takes for them to reach their destination, and their
overall quality of life.
Several
times throughout the 30 days, there were several instances of strangers
approaching the subject and asking if they needed assistance, but most went
about their normal routine without acknowledging the subject. The compounding
symptoms included in the four weeks solicited a strong reaction, positive and
negative, from those around the subject in order to address the emotional and
psychological impacts the disease has on those afflicted. Those demonstrating
the symptoms felt insecure, self-conscious and felt they had higher anxiety
levels compared to their normal routine.
Improvements and
Additional Experiments
Despite the possible advantages
of a test of this indefinite nature, the uncertainty in number of GAA repeats
on the FXN gene is also a cause for a measure of imprecision and ambiguity. The
range of GAA repeats can vary from case to case in those with the disease;
trinucleotide repeats in those with Friedreich’s ataxia can range from 70 to
over 1000 (Ciotti et
al 2004). This setback causes the results to be limited, and could possibly
be corrected by sending in finished PCR products in for genetic sequencing.
This would allow for one to be able to see how many GAA repeats there were,
which would allow for confirmation of successful PCR. If another six weeks were
provided, continual PCR trials could be run, and the most successful run would
be sent in for genetic sequencing. With the sequence of the finished PCR
product, one would be able to see how many GAA repeats there were and how long
the amplified section was, which would either confirm or nullify the supposed
bands. Building upon this, those requesting genetic testing can be left without
information regarding the onset of their disease because of the correlation
between the early onset of symptoms and a higher number of trinucleotide
repeats (Ciotti et
al 2004).
After conducting multiple trials
of PCR and gel electrophoresis, the best result of a band was a smear as
depicted in figure 3. It could have been an incorrect annealing temperature,
degradation of template DNA or primer DNA, or the primers hair pinning.
If another six weeks had been
provided, there would be multiple ways to alter and modify the assay. The first
would be to run many more PCR trials, with multiple cocktails in each run. Each
cocktail would be at a different annealing temperature, done with the
temperature gradient in the automated thermo cycler. This would allow for a
great deal of accuracy in determining the correct annealing temperatures of the
control primers. Additionally, the magnesium chloride concentrations in the
cocktails could be further altered to allow for better amplification, as it is
a co-factor for Taq polymerase. Decreasing amounts of nuclease free H2O
while increasing the amount of PCR buffer could allow for a great concentration
of MgCl2. For each new PCR cocktail that would be run, one could
change the amount of H2O used by 1μl, while at the same time increasing
the amount of buffer by 1μl. This would be done for each new PCR cocktail
run to ensure for a variety of different concentrations. With six additional
weeks provided, the optimal annealing temperature could be found, and magnesium
chloride concentration could constantly be changed to increase the chances of
PCR success. After the control primers show signs of proper annealing to the
desired regions, mutant DNA and heterozygous mutant DNA would be procured and
run with the primers. The results would then be compared and analyzed to
determine the severity of the mutant DNA sample.
Further research may be conducted in
the area of the compound heterozygous FXN mutation that contains the GAA
expansion on one allele and the point mutation affecting the frataxin coding sequence on the other coding for the expression of the
Friedreich’s Ataxia to be active. This point mutation is present in about 5% of
the FA patients and limited studies are available (Campuzano et al 1997). A majority of subjects from
the Campuzano study contained members of their lineage that carried the
mutation or had another ataxic disease. The study found that those in the
lineage who had other muscular dystrophy related diseases eventually had a
descendant who developed Friedreich’s ataxia. Based on this, further
experiments could include a calculation of the percentage of those with
muscular dystrophic disease-producing descendants that developed Friedreich’s
ataxia.
With another six weeks provided,
genetic samples of these descendants could be obtained and tested for how many
GAA repeats they have through PCR and genetic sequencing. By determining the
number of repeats they have, inquiries of whether there is a general
correlation between the number of repeats and risk for muscular dystrophic
diseases. This could provide useful information for those looking for a health
and genetic history of their family and could aid in potential family planning.
Figure 2. Gel electrophoresis product of
amplification of fragment of Rz gene from
bacteriophage lambda. Thermocycling conditions included a 2 minute denaturation phase
at 95°C with 30 cycles of 1 minute at 53°C and a 2 minute elongation phase at
72°C. Lane 1 is Thermo Scientific GeneRuler 1kb Plus
DNA Ladder 75 to 20,000 base pairs. Lanes 2 through 4 are the fragments of Rz gene that were amplified through PCR, and were found to
be 395 base pairs long. The doublets in this figure are noteworthy and may have
arisen from non-specific binds and lower annealing temperatures, as this cycle
did not use a gradient. A solution to this phenomenon may lie in nested PCR.