Identification
of W1282X mutation of CF in Human IB3 Epithelial Cells Using Allele Specific
PCR and Yaku Primers
Team
Watson:
Ted VanAlst,
Jen Beckner, Megan Kechner,
Ryan Furdock
30 Days Experiment:
Trailer: http://www.youtube.com/watch?v=D74BhSAQE9Y&feature=youtu.be
Video: http://www.youtube.com/watch?v=HGwZ1Vpetvg&feature=youtu.be
Abstract
A Yaku approach to primer design with the purpose of developing an allele-specific polymerase chain reaction (ASPCR) assay to detect the nonsense mutation W1282X causing Cystic Fibrosis was followed. This method helped to prevent primer binding to non-target nucleotide sequences (Yaku et al, 2008). Two allele specific Yaku designed primers, one specific for the W1282X mutation and one specific for wild type DNA, in conjunction with a reverse primer complimentary to both alleles were used in PCR with IB3 cell DNA heterozygous for W1282X in the CFTR gene. A Lambda virus experiment and published control primers by Kerem et al. were performed as positive controls. We hypothesized that false-positive results in the PCR diagnosis of the W1282X mutation causing CF would be reduced through the use of primer design with an intentional single base pair mismatch by successfully amplifying a sequence containing the mutation because it would increase PCR stringency and primer discrimination against non-complementary DNA. We found that each set of designed primers amplified a 560 base pair sequence from nucleotides 3953 to 4512 with the PCR protocol used in our controls and optimal annealing temperatures (Kerem et al, 1990). A band corresponding to 560 base pairs was visible in agarose gel electrophoresis when PCR was run with heterozygous template DNA and each set of wild type or mutant primers, supporting the Yaku focus in avoiding false-positive results using intentional mismatches (Yaku et al, 2008). Our assay is significant as it provides a template for development of assays for other genomic mutations and could be useful in expediting the treatment process of patients with W1282X, as it allows for quick diagnosis for specific mutations (Wu et al, 1989). We also investigated the social and psychological implications of CF by wearing surgical masks to simulate interacting with society when seen as someone with a visible, seemingly contagious disease. Experimental trials while wearing the mask yielded significantly (p<0.05) longer time intervals for the zone of inhibition to be entered in two locations as well as significantly (p<0.05) higher Beck anxiety scores in all three locations when compared to control, unmasked trials.
Discussion
Experiment Summary
Cystic fibrosis, the most common
fatal autosomal recessive disorder in Caucasians (Handyside
et al, 1992), is caused by mutation of the CFTR gene on chromosome seven,
causing the absence or malfunction of chloride ion channels in epithelial cells
(Welsh and Smith, 1995). Improper ion movement elicits severe impairment in the
function of the liver, pancreas, digestive system, and lungs by clogging organs
with thick mucus (Rowe et al, 2011). The W1282X mutation is a class I variant
of cystic fibrosis that is especially prevalent among Ashkenazi Jew
populations; it concerns a Guanine to Adenine base substitution of the 3978th
nucleotide causing the conversion of Tryptophan at the 1282nd codon
into a stop codon (Shoshani
et al, 1992). PCR has been deemed an effective tool in diagnosis of a great
number of genetic disorders, a diagnostic utilized by our team to develop an
assay for detecting the W1282X mutation. We hypothesized that false-positive
results in the PCR diagnosis of the W1282X mutation causing CF would be reduced
through the use of primer design with an intentional single base pair mismatch
by successfully amplifying a sequence containing the mutation because it would
increase PCR stringency and primer discrimination against non-complementary
DNA. Our central thesis was that the designed PCR primers would accurately
detect the presence or absence of the W1282X mutation of CFTR and expedite the
treatment process of patients as it would allow for quick diagnosis for the
specific mutation (Wu et al, 1989).
Original
Predictions
Designed forward primers CFTR-F1 and
CFTR-F2 were predicted to be allele specific for wild type and mutant strand
DNA, respectively based on the instituted Yaku primer
design where primers contain a single mismatch for their intended DNA strand
and a double mismatch for their non-intended DNA strand (Yaku
et al, 2008). When used in conjunction with the reverse design primer CFTR-R,
the forward primers were to reveal a 560 base pair amplification of the CFTR
gene. Due to the nonsense point mutation on the 3978th nucleotide
causing the W1282X mutation, forward primers were designed to be sensitive to
this region of the DNA leading to allele specific binding (Shoshani
et al, 1992). A 560 base pair amplification was
expected because the forward primers CFTR-F1 and CFTR-F2 were designed to
anneal from the 3953th base pair of the CFTR gene and the reverse
primer CFTR-R was designed to anneal from the 4513th base on the
CFTR gene (Saiki et al, 1988).
To gain support for our hypothesis,
the pairs of CFTR-F1/CFTR-R and CFTR-F2/CFTR-R were to be tested with
homozygous wild type, homozygous W1282X, and heterozygous wild type and W1282X
DNA. A homozygous wild type DNA template was predicted to only yield bands in
gel electrophoresis when using the CFTR-F1/CFTR-R primers based on the allele
specific design of the forward primer. A homozygous W1282X mutant DNA template
was predicted to only yield bands in gel electrophoresis when using the
CFTR-F2/CFTR-R primers based on the allele specific W1282X mutant sensitive
design of the forward primer. A heterozygous genome of wild type and W1282X
mutant DNA was predicted to reveal a 560 base pair band when analyzed with gel
electrophoresis because both sets of primers would anneal to their respective
alleles. If our designed CFTR-F1, CFTR-F2, and CFTR-R primers followed this
trend, then we could have provided support for our hypothesis of designing
allele specific primers that accurately detect the W1282X mutation.
Over the course of the experiment,
positive control experiments were ran with the Lambda virus Rz
gene and published primers. It was predicted that a 500 base pair amplification
of the Lambda Rz
gene would be achieved when using the 1Rz1F and 1-Rz 1R primers. Once results
from this experiment were obtained, the experiment yielded a positive control
for the PCR cocktail ingredient mixture as well as the PCR protocol. This
experiment could have been reproduced if necessary to ensure the ingredients
used in the PCR cocktail were not contaminated for experiments with our design
primers. For the published primers 20i-5 and 20i-3, a length of approximately 471
base pairs was predicted based on the published research of Kerem
et al, 1990. The published primers provided us with the information that the
region of DNA containing the W1282X mutation location was present in our DNA.
Ultimate Findings
The positive control experiment with
the Lambda virus Rz
gene and 1Rz1F and 1-Rz 1R primers was successful, and from the most distinct
and brightest band, a 505 base pair segment was observed in gel
electrophoresis. With the successful amplification of the Rz
gene, we ensured that the cocktail ingredients and PCR protocol were effective
in PCR. In following experiments, the protocol and cocktail ingredients used
during this effective experiment were utilized. The additional positive control
experiment using published 20i-5 and 20i-3 primers with IB3 cell DNA also
provided beneficial data. The 20i-5 and 20i-3 primers amplified a base pair
segment of 435 base pairs. The presence of a segment of DNA near the predicted
base pair length of 471 supported the findings of the positive control
experiment. The successful amplification of the IB3 cell DNA provides support
that the DNA provided contained the location of the W1282X mutation. Since the
20i-5 and 20i-3 primers were not allele specific for mutant W1282X or wild type
alleles, the only conclusion made from this information was that the region
containing the possible W1282X mutation was present.
Upon the completion of the positive
control experiments with the Lambda Rz gene and the published control primers, the designed CFTR-F1,
CFTR-F2, and CFTR-R primers were tested. CFTR-F1/CFTR-R and CFTR-F2/CFTR-R
primers were tested with IB3 cell DNA known to contain dF508 and W1282X
mutations (Boncoeur et al, 2008). The dF508 mutant
allele was representative of wild type DNA as it did not contain the Guanine to
Adenine substitution on the 3978th base pairing whereas the W1282X
allele contained such a mutation (Shoshani et al,
1992). When CFTR-F1 and CFTR-R were tested with the PCR protocol and with IB3
cell DNA, a band of base pair length 567 was produced in gel electrophoresis. The presence of a band at the expected base pair length provided
support that the designed primers CFTR-F1 and CFTR-R accurately detected wild
type DNA. When CFTR-F2 and CFTR-R were tested with the PCR protocol with
the IB3 cell DNA, an average band of base pair length 540 was produced in gel
electrophoresis. The presence of a band around the expected base pair length
provided support that the designed primers CFTR-F2 and CFTR-R accurately
detected mutant W1282X DNA although there was some nonspecific binding present
that may have required increased annealing temperatures. Since the IB3 cell DNA
was known to be heterozygous for W1282X and wild type DNA, the information
obtained from experimentation followed the original predictions.
Weaknesses In Experimental Design
While the Yaku
Method of primer design can be effective in avoiding pseudo positive results,
there may have been difficulties in creating a suitable environment that
allowed PCR to overcome the intentional base pair mismatch that the CFTR-F1 and
CFTR-F2 primers contained (Yaku et al, 2008). In this
case, a set of traditional primers without a base pair mismatch may have been
optimal in creating allele specific primers.
The primary weakness in the experiment
was the inability to test the design primers with DNA that was homozygous for
wild type or mutant W1282X DNA. Although the design primers were successful in
amplifying segments of DNA at the expected base pair length, it was uncertain
whether the primers were in fact allele specific. Testing CFTR-F1 and CFTR-F2
primers with the reverse primer CFTR-R with DNA homozygous for wild type or
mutant W1282X DNA would have provided further support for the designed allele
specific primers. This additional information could have aided in affirming
that the primers did not both anneal to the same allele or that they each
annealed to both alleles of DNA.
Unmasking Cystic
Fibrosis: A Socio-Psychological Approach
In addition to the PCR assay, a
sociological experiment was to be conducted in which the socio-psychological
implications of living with cystic fibrosis were explored. The experiment
focused on the influence of the zone of inhibition created by wearing a
surgical mask in social situations. We hypothesized that the visible symptoms
of cystic fibrosis would be perceived in a negative way by members of society
because people may fear that it is contagious; we also hypothesized that
someone subjected to being perceived in such a way would experience more anxiety
in knowing they were being negatively judged (Beck et al, 1988). A t-test was
utilized to test the statistical significance of time differentiation between
masked and unmasked trials as well as testing the anxiety levels based on the
Beck Anxiety Inventory from masked to unmasked trials (Beck et al, 1988), (McCluskey et al, 2007). A p-value less than 0.05 provided
statistically significant results between masked and unmasked trials.
Experiments performed in residence hall cafeterias produced a statistically
significant (p=0.00078) for zone of inhibition times as well as significant
(p=0.00344) results between masked and unmasked anxiety scores. Experiments
performed in MSU lecture halls also produced significant (p=0.008193) results
for zone of inhibition times as well as significant (p<0.0001) results for
anxiety scores. Lastly, experiments performed on the CATA buses produced
insignificant (p=0.549) results for zone of inhibition times while producing
significant (p=0.0009) results for anxiety scores. This evidence supported our
two hypotheses that individuals perceived to have visible symptoms of cystic
fibrosis would be avoided in social situations, as in the cafeteria and
classroom settings, and that individuals being perceived in such a way would
have increased anxiety levels in any circumstance. The bus trials may have been
insignificant due to a high concentration of people in limited space, as well
as individuals knowing they would only be exposed to test subjects for a short
amount of time.
Future Directions
There
was significant streaking observed in the 1kb+ ladder used in gel
electrophoresis during the designed primers section of the assay. For these
gels, 1µg/mL Invitrogen ladder was used, however 0.1µg/mL
1kb+ and 100bp ladders were used in the control sections of the experiment.
Although lesser volumes (3μl)
of the more concentrated ladder were used, error may have resulted from the
pipetting of too great an amount of ladder into its designated wells. This
problem might be resolved by returning to using ladder of lower concentration,
or by experimenting with lesser volumes of the more concentrated ladder.
Imperfect gels, evidenced by the
streaking and bowing of ladders, may have also been due to our group’s limited
time in the laboratory (4hrs/week). Our need to make gels quickly often forced
us to speed up the cooling of our gels by refrigeration and water cooling. This
may have interfered with the polymerization of the agarose
matrix in some instances; this problem could be remedied by allowing the gel to
cool and solidify without outside input.
In order to provide greater support for the diagnostic assay involving W1282X allele specific PCR, the designed primers CFTR-F1, CFTR-F2, and CFTR-R could be run in PCR with template DNA that is known to lack the W1282X mutation. Upon analysis in gel electrophoresis, it is predicted that bands 560 base pairs in length will be visible only when CFTR-F1 and CFTR-R are placed in the cocktail with this homozygous non-W1282X DNA because of the inability of Yaku-designed primers to anneal to non-target DNA (Yaku et al, 2008). An identical follow up test could be conducted with homozygous W1282X DNA; bands will only be visible when CFTR-F2 and CFTR-R are placed in reaction cocktail with homozygous W1282X DNA.
Once these results are obtained, this assay’s accuracy in detection of W1282X mutation of CF in unknown DNA could be tested. A single blind study could be conducted, in which those who are following the assay have no knowledge of the nature of the DNA being tested (Wu et al, 1988). This assay could be considered useful and effective in the medical field and others if it earns a score at or near 100% accuracy in this single blind study.
In the sociological experiment performed, significant results (p<.05) were obtained. Although many precautions were taken to reduce influence of outside variables, differences in clothing were only taken into account as a variable in the bus trials. In order to provide greater support for our results, the cafeteria and classroom trials could be repeated with the experimenters wearing the same clothing in each trial.
Figures
5000 400 650 1650 2000 1Kb Plus Ladder 1Kb Plus Ladder 51.9˚C 53.8˚C 56.1˚C 58.0˚C 59.2˚C 60.0˚C 50.7˚C 50.0˚C
Figure 5- PCR Amplification of IB3 Cells
Using CFTR-F2 (Mutant) and CFTR-R Designed Primers. (a) IB3 cells containing the W1282X and ∆F508 Cystic Fibrosis
mutations were analyzed with PCR. The region containing the W1282X mutation was
amplified by the CFTR-F2 forward primer (5'-TTCAATAACTTTGCAACAGGGA-3’) and CFTR-R
reverse primer (5'-TTTGTGACCAAATCAGAGTGAC-3‘). Distinct bands were found for
varying annealing temperatures for the same PCR cocktail. Three 150µl master
mixes were made from 114µl nuclease free water, 21µl 10X PCR buffer (15 mM MgCl2, 500 mM KCl, and 100 mM Tris-HCl, pH 8.3), 3µl of 10mM deoxyribonucleotide
triphosphates (dNTPs), 3µl
of 100mM CFTR-F2 primer, 3µl of 100mM CFTR-R primer, 3µl IB3 cell DNA, and
1.5µl taq polymerase. These three master mixes were
split into 50µl aliquots which were each run in PCR with varying annealing
temperatures. PCR began with an initial denaturing phase of 95˚C for 3
minutes, and was followed by 25 cycles of denaturing, annealing and extension.
The denaturing phase was 95˚C for 30 seconds, the annealing phase
temperatures were unique to each 50µl aliquot and were 50.0˚C,
50.7˚C, 51.9˚C, 53.8˚C, 56.1˚C, 58.0˚C, 59.2˚C,
and 60.0˚C, run for 30 seconds, and the extension phase was 72˚C for
1 minute. After the completion of the 25 cycles, a final extension phase of
72˚C ensued for 5 minutes. A 0.8% agarose gel
was made using 0.8g agarose, 100ml TBE (Tris/Borate/EDTA), and 2µl of GloGreen
dye and was used to analyze the PCR products. Wells 1 and 11 contained 2 µl 1Kb
Plus Ladder and 3µl of loading dye. Wells 2-10
contained 3µl of loading dye and 7µl of PCR product and were arranged in
ascending order so that the lowest annealing temperature sample was in well 2
and the highest in well 10. The gel was run at 120V for a period of 35 minutes.
The gel was observed under an ultraviolet light and distinct DNA bands were
present near the 500 and 600 base pair markers of the DNA ladders. Looking at
the lower temperature wells there appeared to be non-specific binding present,
but in well 5 there was a bright, clear band, supporting the annealing temperature
of this would support that the higher
annealing temperatures around 59.2˚C and 60.0˚C are nearer the
optimal annealing temperature 53.8˚C for the CFTR-2F and CFTR-R primers.(b) Utilizing the 1Kb Plus base pair
ladder, a semi-logarithmic plot of Molecular size (bp)
vs migration distance (cm) was used to analyze the
DNA bands. Using an exponential regression curve, the equation y=38514e
(-.908x) was derived from the plotted points. By measuring the distance
the PCR produced bands are from the loading wells, an accurate band length was
determined for each visible band. The band distance for well 5 was 4.7 cm corresponding
to a base pair length of 540 bp. An R2 value of 0.9754 for the
exponential regression trend line was close to a perfect fit of 1 for the trend
line, however, it still included some error.
1Kb P
