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Figure 1: Amplified PCR product from IB3 and S9 cell lines with varying annealing temperatures and primer concentrations.
Thermocycling conditions included a 5-minute denaturation at 94 ºC with 30 cycles of 30 s at 94 ºC, 30 s at 46 or 49 ºC,
and 60 s at 72 ºC, with a final elongation phase at 72 ºC. Lanes 4 and 8 for both cell lines show distinct bands for the
target sequence length of 1056 near 1018 base pairs on 1 kb ladder (lanes 1 and 12). IB3 lanes 3,4,5,7,8,9 and S9 lines 3,4,7,and
8 show non-specific binding. All other lanes show no results.
Figure 2: Amplified PCR product from S9 cell line searching for R553X mutation by varying MgCl2 and primer concentrations.
Thermocycling conditions included a 5-minute denaturation at 94 ºC with 30 cycles of 30 s at 94 ºC, 30 s at 46 or 44 ºC, and
60 s at 72 ºC, with a final elongation phase at 72 ºC. Lanes 4, 5 and 9 show wild-type bands for the target sequence length
of 1056 base pairs near 1018 on 1 kb ladder (lane 11). Lanes with bands showed non-specific binding. Lane 7 shows a faint
band past 506 base pairs. All other lanes show no result
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Discussion
Experiment Summary
Cystic fibrosis, the most common autosomal recessive disease in Caucasians (Aznarez et al, 2007), has dramatic effects
on multiple organs, including the lungs, pancreas, intestines, and liver (Welsh and Smith, 1995), due to mutations of the
CFTR gene on chromosome seven, causing defects in sodium and chloride transport in epithelial cells (Aznarez et al, 2007).
The R553X mutation is a specific variation of cystic fibrosis, involving a single base pair substitution at the 1789th base
pair in the 553rd amino acid, from cytosine to thymine (Hull et al, 1993). The subsequent change from the amino acid arginine
to a premature stop codon causes early truncation of the CFTR protein, thus altering the folding sequence (Gambardella et
al. 2006). Although PCR has been proven effective for diagnosing genetic disorders such as cystic fibrosis (O'Leary et al,
1997), the question we are addressing is whether or not a PCR test can be designed to identify this specific mutation. We
hypothesized that allele specific primers and a single base pair mismatch could be used to develop an accurate diagnostic
test for patients with the R553X mutation using experimentally determined optimal conditions of PCR in terms of annealing
temperature, primer concentration, and salt concentration.
In addition to primer design, we bridged the gap between laboratory experiments and the sociology behind diagnosing
genetic diseases by surveying student opinion on genetic screening and the effects of genetic diseases on the human race in
the long run. In recent years modern medicine has extended the life expectancy of people with cystic fibrosis allowing those
affected to live to childbearing age (Ratjen 2008), meaning CF genes are more likely to be passed down to future generations.
Samples were taken from Lyman Briggs, James Madison, the College of Natural Science, and general university students. It was
hypothesized that Lyman Briggs students would be more supportive of genetic testing due in part to their background in required
integrated studies and their greater understanding of how the frequency of a genetic disease could impact a gene pool (Singer
et al, 2008).
Original Predictions
By amplifying DNA from IB3 human bronchial epithelial cells from a CF patient and S9 epithelial cells from a leukemia
patient via allele specific PCR, the length of the amplified DNA was interpreted through gel electrophoresis to show the presence
or absence of the R553X mutation. Two different forward primers, Fprimer1 and Fprimer2, were designed to discriminate between
the wild-type and mutant CFTR genes through allele specificity based on a single base pair mismatch on the 3' end. The mismatch
was positioned on the 3' end of the primers to more effectively reduce the amplified product by decreasing DNA polymerase
and dNTP binding efficiency (Yaku et al, 2008). Successful annealing of the primers and the subsequent extension phase was
hypothesized to result in a band of 1,056 base pairs, thus indicating a positive test. The lack of a band was hypothesized
to indicate a disruption in the extension phase due to the single base pair mismatch (Chavanas et al, 1996). A homozygous
wild-type genotype was expected to show a band of 1,056 base pairs when using Fprimer1 and show no band when using Fprimer2.
In contrast, a homozygous mutant genotype was expected to show a band of 1,056 base pairs when using Fprimer2 and show no
band when using Fprimer1. Lastly for heterozygous genotypes, faint bands 1,056 base pairs long were expected to appear in
both tests, using either Fprimer1 or Fprimer2 due to the replication of both genotypes during PCR, causing neither set of
forward primers to completely discriminate against the specific mutation site (Chavanas et al, 1996).
Results and Ultimate Findings
In order to determine optimal PCR conditions, multiple experimental trials were run with adjustments in DNA concentration
and primer concentration. In addition, multiple experiments were run to establish the optimal annealing temperature, which
can directly affect the annealing rates of designed primers (Elnifro et al, 2000) and magnesium chloride (MgCl2) concentration
to alter magnesium ion concentrations, which directly affect DNA polymerase activity in PCR (Ignatov et al, 2002). The optimal
annealing temperature was determined to be 46ºC based off of the calculated primer melting temperatures (see Methods section)
and which annealing temperature resulted in the clearest bands and least amount of nonspecific binding. A higher concentration
(10x) of primer was found to be more effective for amplifying DNA from S9 epithelial cells. This could have been due to the
shorter incubation time (1 minute) used during DNA extraction, resulting in a lower concentration of DNA. As a result, a
higher concentration of primer may have been necessary to increase the probability of annealing. In contrast, IB3 epithelial
cells were incubated for 20 minutes, possibly resulting in a higher concentration of DNA extracted, exhibited excessive nonspecific
binding when 10x primer was used. A PCR test run using IB3 cells and primers from a fellow research group studying the same
mutation showed that 1x primer concentration yielded the clearest wild-type IB3 bands obtained with a relatively small amount
of non-specific binding.
The bands of the expected length obtained support our hypothesis of using allele specific primers as a method of diagnosing
the R553X mutation. However, our hypothesis was refuted by the lack of accuracy experienced due to high instances of smearing,
possibly due to nonspecific binding. In early gels, trials using the mutant seeking primer and S9 stock cells yielded mutant
bands of the proper length (about 1,056 base pairs) indicating a possible mutant genotype (Figures 1, 2, and 3), however,
later gels that were run with varying levels of MgCl2 and primer concentration showed only very slight smearing in samples
made with the mutant seeking primer and no positive band (Figure 5). This discrepancy between the results regarding the genotype
of the S9 epithelial cells may be a result of finding the more optimal conditions for PCR, which could have reduced the nonspecific
binding that caused early pseudopositive mutant bands. However, it may also be a reflection of the ineffectiveness of our
allele-specific primers. Later research revealed that A/C (primer/template) mismatches on the 3' end, which is the mismatch
by which our mutant seeking primer functions, are predisposed to allowing PCR amplification, resulting in false positives
(Yaku et al, 2008). Another possible source of error in experimental procedure that could explain excessive nonspecific binding
is the contamination of materials such as pipette tips or samples.
In addition to pseudopositive bands observed using the mutant seeking primer, a consistent band at about 400 base pairs
was observed in various gels using both mutant and wild-type seeking primers (Figures 1-5). The consistency with which the
band appears could mean that there exists a sequence on the CFTR gene that our primers are consistently able to bind to, resulting
in a successful extension and amplification. Although the band shows when using either the mutant seeking primer or the wild-type
seeking primer, it was found to be more prominent when using the mutant seeking primer (Figure 5), which may be an indication
that it is the forward primers that are binding to an alternative place on the gene and the single base pair difference between
Fprimer1 and Fprimer2 enables Fprimer2 to more effectively bind to that alternative site.
Results obtained from the sociology research refuted our hypothesis that Lyman Briggs students would be more likely
to support genetic testing. According to statistical analysis of responses from Question 1 of the survey (see Methods section),
the test groups showed no statistical differences in their support or opposition of genetic testing. However, statistical
differences calculated for responses from Question 8 showed that Lyman Briggs students found integrated studies courses to
be more useful in formulating an understanding of issues behind genetic testing as compared to all three other survey test
groups. These results somewhat support our hypothesis because they show that coursework and previous knowledge of genetics
has an effect on an individual's opinions of genetic testing.
Future Direction
To improve our experiment, new primers could be designed to have a higher G/C concentration to enhance binding efficiency
during the annealing phase (Elnifro et al, 2000). The accuracy of our existing primers could be more precisely tested if DNA
known to have the R553X mutated genotype was obtained for testing so the effectiveness of the single base pair mismatch of
the Fprimer1 with mutant R553X CFTR could be tested in addition to the use of Fprimer2 to properly amplify the DNA. Further
research has indicated that primers which are allele specific on the second nucleotide from the 3' end and have an additional
single base pair mismatch to the original DNA template on the third nucleotide from the 3' exhibit a lower chance for a false
positive (Yaku et al, 2008), thus providing a possible future protocol for more effective primer design.
Further experiments involving restriction enzymes and DNA sequencing could be used to ensure that bands of the correct
length that were obtained reflect the amplification of the correct targeted section of DNA. Restriction enzymes could be used
to splice the amplified DNA at specific sequences (O'Leary et al, 1997) and gel electrophoresis could be used to analyze the
resulting bands. For the specific DNA amplified in our experiment, the restriction enzyme BamHI could be used to splice the
1,056 base pair amplified DNA, at the 5'-G/GATCC-3' sequence (Mones et al, 2007), into two separate bands of 530 and 521 base
pairs.
Possible future protocols for improving the sociology research could include obtaining a larger samples size of students
at Michigan State University, especially those in James Madison College. These samples could be obtained by asking more James
Madison professors to distribute the survey URL to their students and by utilizing ANGEL, MSU's online course communication
system, which proved effective for obtaining samples from students in Lyman Briggs College and the College of Natural Science.
To take the research even further, a comparison could be done not only between residential colleges at MSU, but also between
different universities.
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