Comparison of Primer Design to
Diagnose the V232D Mutation
of CFTR Using PCR and Gel
Electrophoresis
Danielle Hohman, Oren Lerner, Scott
Nolan, Grace Kuza, and Zakia Zaman
LB 145 Cell and Molecular Biology
Tuesday 10:20 AM
Davin Hami and Ali Kadouh
12/4/2018
Abstract
There are over 1,700 mutations of
the CFTR gene that lead to the autosomal recessive disease cystic fibrosis (CF)
(Lorenzo et al., 2018). Of these, the V232D mutation is a
rare condition caused by a single nucleotide polymorphism of the 232nd amino
acid. At the mutation site, a T→A nucleotide change causes valine (GTC),
to turn into aspartic acid (GAC). This promotes the formation of non-native
hydrogen bonds which lead to a misfolding of the CFTR protein (Choi et al., 2004). To diagnose the V232D mutation of CFTR, an array of
polymerase chain reaction (PCR) tests were designed and analyzed using gel
electrophoresis. Preliminary control experiments were conducted by analyzing a
known segment of the E. Coli genome and purifying wild-type DNA from somatic
buccal cells using chelex resin. Additionally, a sample of V232D DNA was
acquired from The Hospital for Sick Children in Toronto, Ontario. To analyze
the mutant DNA, four primers were designed to compare amplification versus
nucleotide placement. Of the four primers, one was a wild-type seeking forward
primer, one was a common reverse primer, and two were mutant seeking forward
primers. We hypothesized that a primer designed with a nucleotide mismatch on
the second annealing site from the 3’ end (55,399 bp to 55,423 bp) will be more
effective at diagnosing the V232D mutation of CF than a primer with a mismatch
on the third annealing site from the 3’ end (55,400 bp to 55,422 bp) because of
steric strain limiting false positive results. To test this, PCR was conducted
with a thermocycler to amplify the sequences. The amplified DNA was then
analyzed using gel electrophoresis to determine the primer efficiency on each
sequence. The final analysis yielded data to support the function of our mutant
seeking primers on mutant DNA. On the other hand, not enough data was collected
to support the relationship between primer design and diagnostic efficiency.
Therefore, we reject our hypothesis, prompting further studies to investigate
mismatched nucleotide placement versus annealing and polymerization.
Discussion
Experiment
Summary
Cystic fibrosis (CF) is the most
common autosomal recessive disease in Caucasians (Ernst, 2011). This lethal
genetic disease presents itself with multisystemic failure mainly affecting the
respiratory system, gastrointestinal tract and reproductive organs (Welsh,
2001). The genetic base for the disease is due to the alteration of the cystic
fibrosis transmembrane conductance regulator (CFTR) gene which primarily
encodes for a chloride channel protein essential for ion transport (Rommens,
1989). However, there are large numbers of CF-related mutations where the
disease is caused by a misfolding in the chloride channel (Caldwell, 2011).
With CF, alteration or loss of function of the protein leads to an imbalance of
ion exchange, resulting in dehydration of the mucous secretion in many organs,
most notably within the respiratory tract organs (Welsh, 2001). V232D is a
mutation of the CFTR gene that exhibits defective folding and trafficking
(Caldwell, 2011). The CFTR gene consists of 27 exons, spanning more than 250
thousands base pairs, located on exon 6a is the V232D mutation. This genetic
mutation is a change from a Valine (GTC) to Aspartic Acid (GAC) within exon 6a,
nucleotide 827, in the protein sequence at the 232nd Amino Acid. The folding
defect caused by the V232D mutation appears to be due to the introduction of a
charged residue into a region of CFTR that is embedded in the lipid bilayer of
the endoplasmic reticulum (ER) membrane (Rath, 2009). The V232D mutation is
considered to cause less extreme symptoms; however mild symptoms can
nonetheless cause significant damage to livelihood and life-expectancy
(Fernández-Lorenzo, 2018). Following the study of the mutant V232D, it is
hypothesized that a primer designed with a nucleotide mismatch on the second
annealing site from the 3’ end (55,399 bp to 55,423 bp) will be more effective
at diagnosing the V232D mutation of CF than a primer with a mismatch on the
third annealing site from the 3’ end (55,400 bp to 55,422 bp) because of steric
strain limiting false positive results. Our central thesis is that by using our
custom made primers and PCR protocol, the V232D mutation will effectively and
accurately be identified, leading to advances in the medical field because
doctors will be able to diagnose this mutation in patients more
efficiently.
Original
Predictions
We used four different primers to
conduct our experiment. These primers consisted of a universal reverse primer,
which was the same for both wild-type and mutant samples, the wild-type forward
primer and two designed reverse mutant primer (Kwok, 1994). The first mutant
forward primer has a variation accounting for the single change in the
nucleotide sequence found two nucleotides in near the 3’ end (F2), whereas the
second mutant forward primer has the nucleotide mismatch on the third
nucleotide in (F3). The forward wild-type primer (F1) was used to distinguish
between the CFTR gene respective mutant and wild-type DNA samples in accordance
with the single nucleotide mismatch. Further, we predicted that after running
the gel for the mutant-type DNA using the different forward primers there would
be no band using the wild-type designed, a band of 1046 bp using F1, and a band
of 1045 bp using F2 because the mismatch does not allow the wild-type primer to
bind to mutant DNA (Sommer, 1989). We also predicted that when the wild-type
DNA was run, there would be a band of 1046 bp using the wild-type primer, no band
using F2, and a band of 1045 bp using F3 because the mismatch in F3 is shifted
in far enough that Taq Polymerase can bind to the 3’ end and still amplify the
DNA due to steric strain, whereas when using F2, Taq is not able to bind to the
strand is not amplified (Sommer, 1989).
Results and
Findings
The optimal annealing temperature
was found to be 53℃ which is 2℃ less than the lowest melting
temperature, 55℃. This allowed proper denaturing and annealing without
melting the primers themselves (Fernández-Lorenzo, 2009). The concentrations of
our DNA and primers in addition to our annealing temperature had minute
adjustments made over many trials to ensure the best combination was utilized
for the assessed PCR reaction. Once electrophoresis was completed, the gel was
assessed under UV light to recognize the DNA separation. The first trial was
not successful in producing bands and was a result of improper gel design and
PCR cocktail. The agarose solution lacked SYBR safe which prevented the viewing
of any possible bands. Note of the error was made and the gel was then soaked
in a solution of 
of 5X TBE and 6
SYBR dye, hypothesizing that it would absorb
some of the dye and allow visible bands. Only the DNA ladder became present
from this step and acts as a control for the overall experiment by verifying
the gel ratio was adequate. The expected band was not present due to the lack
of Mg+2 in the PCR cocktail, which is an essential cofactor that
increases the productivity of Taq polymerase (Fernández-Lorenzo, 2009).
Amplification of E. Coli bacteria
occurred using 8F and 1512R primers in a PCR cocktail that accounted for MgCl2
and it was run in a gel alongside a kb+ ladder. A band of 1504 bp was expected
and as seen in the semi-log graph in Figure 2, a band of 1507 bp was received.
It was not exactly 1504 due to a potential well morph error altering the shape
slightly making it seem 3 bp longer in the graph (McCauley, 2018). The bands
appeared very fluorescent due to large amounts of amplified DNA and a higher
concentration of the SYBR safe. Smears in the gel are a result of the use of an
old gel (Hellman, 2007). The E. Coli test acted as a positive control for the overall
experiment because it produced a band of the expected base pair length which
confirmed our PCR cocktail. Amplification of genomic buccal cells using
published primers obtained from Ana E. Fernández-Lorenzo of the Department of
Pediatrics at Hospital Teresa Herrera, Coruña, Spain. A band of 385 bp was
expected and as seen in the semi-log in Figure 4, a band of 387 bp was
received. It was slightly longer than the expected due to well morph error
which was seen during the E. Coli experiment (McCauley, 2018). It was noticed
that the band for the genomic is faint. This is most likely a result of not
enough genomic from the purified solution, therefore less was amplified when
PCR was conducted (Hellman, 2007). This served as a method control because there was
enough genomic DNA purified from it that produced a band after amplification.
Figure 3 is the purified genomic before amplification. The genomic DNA strength
was tested in two ways: loading it into wells and then using UV light to see it
fluoresce and testing its DNA and RNA content using the spectrophotometer as
well as running genomic DNA in a gel and seeing how well the wells fluoresce.
Amplification of our wild-type
genomic DNA with the three different forward primers occurred. When amplified
with F1 a band found at 1083 bp was present, the expected was 1046 bp. The band
very faint due to the lack of genomic wild-type DNA in our sample that bound
with Taq. The DNA that did not bind with Taq stayed in the well which is why we
notice well two fluoresce. Neither the F2 or F3 primers, both which were
complementary to the mutant DNA, annealed to the wild-type DNA which is why
lanes three and four lack bands. There is a noticeable streaking present which
is a result of too much DNA loaded into the wells (Hellman, 2007). From the data we received, we refute
our original hypothesis that F3 would bind to our wild-type DNA giving a false
positive due to the lack of bands seen in the gel when run with wild-type DNA.
All three forward primers yielded a band of 877 bp when amplified with the
mutant DNA. It is believed that the cocktail for the F1 and mutant DNA might
have been cross-contaminated with the small amounts of F2/F3 primers the
addition of mutant DNA because the same pipette tip was used in each solution
which would explain the faint band. Both F2 and F3 primers were expected to
yield bands because they were complements of mutant DNA. Even though the
received band was 877 bp it was expected to be 1046 bp for F2 and 1046 bp for
F3. They were shorter than expected due to potential DNA degradation because
they were kept at room temperature for a lengthy duration of time before
refrigerated.
Potential cross-contamination of
wild-type DNA and mutant DNA could have occurred since our lab setup did not
follow sterile protocol. The calibration of the micropipettes were off and
varied between each individual due to incorrect handling. This gave incorrect
ratios in the PCR cocktail which would explain why some of the bands appear to
be faint (Adey, 2014). The genomic purification process does not guarantee
purified DNA. Due to the lack of precise filtering out of proteins, RNA, and
other macromolecules which were still present and could have attributed to
incorrect results found in the gels (Glasel, 1995). If there was more time to continue
this experiment, a better wild-type genomic purification would occur as well as
more precise measurements of each ingredient found within a PCR cocktail to
ensure that the results would be more accurate.
Future Directions
Upon moving forward and analyzing
any experimental errors, we noticed a recurring problem of streaking amongst
the bands. To specify, streaking was observed when viewing results from the
E.Coli control, wild-type DNA with F2 primer as well as F3 primer, and
mutant-type DNA with all three proposed primers. Streaking was an indication of
excess SYBR safe, independent of the amount of DNA used to load the wells.
Originally, 6 was used to illuminate the bands. Because we
worked with mini gels instead of larger gels, this was 1too much of SYBR safe (Li-Cor,
2011). Due to multiple gels with streaking, it would be beneficial to decrease
the original amount of SYBR safe by
, to 5, which can reduce streaks and
result in a clearer gel. Other complications can be seen on the gel in Figure
4., where dark, cloudy spots can be spotted towards the bottom of the gel of
the wild-type. These dark spots indicated that there was a high concentration
of the blue loading dye as well as the DNA in the well. The loading dye is used
to assemble the migration distance on a gel. To eliminate the dark spots, the
gel can be “destained” by soaking in distilled water. It is important to be
careful with the duration in which the gel is soaked, as prolonged exposure can
interfere with the staining of the bands (Hervieu, 1997). With furthermore
examination of all agarose gels under UV light, specks continuously came up, as
seen in Figures 2, 3, 4, and 5 obtained from experimentation. These specks
often result from dust particles on the gel. The specks result from the
preparation process, looking specifically at cleaning/drying methods used
during the preparation of the gel electrophoresis chamber and comb. Over the
duration of this experiment, brown paper towel was used to clean any equipment,
giving a large possibility for dust or particles to stick to the
electrophoresis chamber. A switch to lint-free tissue paper can minimize the
specks present in an observed gel (Westermeier, 2005).
Figures
Results of
PCR with designed primers on wild-type DNA and mutant DNA. Each PCR cocktail contained the
universal reverse primer 5’-GTAAATGCCTCCTATGTGCCAGAC-3’. For WT+F1 and M+F1 the
forward primer 5’-TGTGGACTTGGTTTCCTGATAGTC-3’ was used. For WT+F2 and M+F2
forward primer 5’-TGTGGACTTGGTTTCCTGATAGAC-3’ was used. For WT+F3 and M+F3 the
forward primer 5’-GTGGACTTGGTTTCCTGATAGACC-3’ was used. 4 of the 100bp ladder and 13
of each PCR product were pipetted
into the wells of an agarose gel. The gel was run for 17 minutes at 300 volts. Both 100bp
ladders appeared distorted due to prematurely pulling out the well comb. The
WT+F1 produced a band of 1083 base pairs and it turned out very light.
Additionally, some genomic DNA was left in the WT+F1 well. The expected length
was 1046 base pairs, but 1083 was sufficient enough that we deemed this result
correct. The band was very light because there wasn’t a high volume of DNA that
traveled, and some stayed in the well because there was extra genomic DNA in
the mixture. The WT+F2 and WT+F3 did not yield any results. The M+F1, M+F2 and
M+F3 all resulted in bands with lengths of 877 base pairs. The M+F1 is lighter
than the other two bands and they all have a bit of streaking. M+F1 was not
expected to work at all, M+F2 was expected to have 1045 bp and M+F3was expected
to have 1046 bp. The mutant sample bands were shorter due to potential
DNA degradation because they were kept at room temperature for a lengthy
duration of time before refrigerated. There was also possible
cross-contamination between the mutant and wild-type DNA due to unsterile
practices in the lab, such as not switching out pipette tips