Detection
of the R117H Mutation in the Human CFTR Genome for Mild Cystic Fibrosis
Utilizing Allele Specific PCR
By:
Zoe Buszka, Kalin Bayes, and Danielle Guist
LB 145
Cell and Molecular Biology
Lab
Time:Tuesday and Thursday 10:20-12:10pm
Matt
VanDer and Allison Vlk
10/10/2017
WEBSITE
Title
page written by: B945
Revised
by: B980
Finalized
by B925
Abstract
Written by: B980
Revised by: B925
Finalized by: B945
On the
seventh chromosome of the human genome resides a mutation in the CFTR gene
known as R117H; at the 117th amino acid a guanine is altered to an adenine. It
has been found that 4 of 18 cystic fibrosis patients tested show signs of
having this mutation (Gervais et al, 1993), however it is not routinely
screened for. Developing an accurate, inexpensive test for this mutation allows
for individuals to be tested for the mutation of interest and treated quickly.
Polymerase chain reaction was used to amplify the target DNA, containing the
R117H mutation, with designed primers.It was hypothesized that the designed
primers for the R117H mutation that causes cystic fibrosis will bind to the
nucleotides, at 56.5¼C, of the mutant DNA, but not to the wild type DNA. The
resulting amplified DNA was then analyzed using agarose gel electrophoresis.
The agarose gel showed three band lengths: one of 212 base pairs and one of 432
base pairs for DNA containing the R117H mutation, while one band of 307 base
pairs was present containing the wild-type DNA. This allowed for the
identification of an individual who possesses this specific mutation because
the primers will bind to and replicate the mutant DNA. We predicted that three
designed primers would detect the R117H mutation that leads to cystic fibrosis
through genetic analysis because PCR is an effective method at amplifying
target DNA for genetic mutations (Balogh et al 2004).
Discussion
Written by: B980
Revised by: B925
Finalized by B945
Experimental
Summary
Cystic
fibrosis is a recessive genetic mutation that causes the CFTR chloride channels
within cells to operate poorly, if at all (Ying-chun, 2016). Patients with
cystic fibrosis often have large amounts of secretions coming from their lungs,
pancreas, and intestines that create a perfect environment for bacteria to
culture, in addition to causing breathing difficulties (Rowe et al., 2005). At
the 117th amino acid of the CFTR gene a guanine is altered to an adenine, which
results in the R117H mutation. The R117H mutation can either extend the opening
or closing time of the chloride channels present in the CFTR gene. We are
performing this experiment to answer the question of whether or not a PCR test
will be able to amplify DNA samples well enough to determine if an individual
has the R117H mutation early on or if the individual is a carrier of the gene.
We hypothesize that the designed primers for the R117H mutation that causes
Cystic Fibrosis will bind to the nucleotides of the targeted mutation but not
in wild type DNA because the primers were created correctly and the annealing
temperature was carefully calculated. This will allow the mutation to become
two visible bands when ran through a gel electrophoresis and causing the wild
type DNA to form a single band.
It
would be beneficial to create an assay that tests for the top 10 most common
mutations causing cystic fibrosis. This can be done by creating a set of
primers that amplify most of the mutations linked to cystic fibrosis to be
further analyzed through restriction enzymes which cuts at different points in
DNA that contains the mutations. This would provide the different band widths
to identify individual mutations of cystic fibrosis in the human genome. This
would be a beneficial test that would tell parents if they were positive for
one of the mutations and how likely it would be for a coupleÕs child to develop
cystic fibrosis if they decided to have one.
Original
Predictions
The
results of the experiment would have shown that the designed primers did
successfully bind to the DNA strand and amplified the target DNA because there
was a band present of 212 base pairs and 432 base pairs, in the agarose gel,
that would be compatible to the R117H mutation band in the control experiment
(Shrimpton, 2012). This means that our data supported our hypothesis and that
our results would have aligned with previously published research. PCR is a
common method for replicating DNA and assisting in performing genetic
screenings successfully for point specific mutations. Our research was
performed with the same basic steps as other published papers; the major
difference was that we designed our primers to include DNA bands with longer
lengths so they would be more prominent in gel electrophoresis.
While
similar to other published papers, this research proves it is possible for the
R117H mutation to be detected in patients if they have a genetic screening
performed on them. It is rare that R117H is tested for due to the disease being
unrecognized by doctors until significant symptoms become prevalent, however it
should be because the frequency in those with cystic fibrosis is high (Gervais
et al, 1993). These experimental results indicate that the assay should be
performed successfully with no harm to the patient and no hassle with the
analysis. This test can also help expecting parents to test their genomes for
the R117H genetic mutation as early as possible (Malone et al, 1999).
Results
and Ultimate Findings
In
order to determine optimal PCR conditions for the R117H mutation, multiple
experimental trials were run with adjustments in DNA concentration and
magnesium sulfate concentration. In addition, these experimental trials were
run to establish the optimal annealing temperature, which can directly affect
the annealing rates of the published and designed primers. The optimal
annealing temperature for our published primers was determined to be 56.5 ¼C
(see Methods section). The optimal concentration of magnesium sulfate was 1.5
ul. Taq polymerase is a magnesium-dependent enzyme, so determining the optimum
concentration to use was critical for the success of the PCR reaction
(Markoulatos, 2002). After the optimal PCR conditions were determined, a
clearly defined band of 307 base pairs long that contained the published primers
for the wild-type DNA and a clearly defined 1 Kb plus ladder were produced.
This is in support of our hypothesis that that the published primers would
anneal properly to the wild-type DNA to form a single band of 307 base pairs.
Future
Directions
If research
was to resume, the PCR assay containing mutant DNA would be performed. The
designed primers would be ordered and the mutant DNA obtained containing the
R117H mutation. The optimal DNA concentration, magnesium sulfate concentration,
and annealing temperature, for the mutant DNA and designed primers, would be
found through multiple experimental trials. Performing a PCR assay containing
the mutant DNA would further prove our hypothesis that our designed primers
would anneal correctly producing two bands of 212 base pairs and 432 base
pairs. This would provide evidence that using PCR is an effective assay for
detecting the R117H mutation for genetic mutation screening in cystic fibrosis
patients.
An
experimental weakness that was encountered was properly preparing the PCR in
order to replicate the target DNA. Creating the proper ratio of all reagents
and the concentration is vital for the target DNA for PCR. Depending on the
primer length different concentrations and binding agents were used to amplify
DNA. Magnesium is an example of a reagent that is necessary for PCR to properly
replicate DNA (Barlett, 2003). Another analysis of experimental flaws was
inserting the samples into wells. Often, the DNA was allowed to exist in the
saline solution, or a hole was poked into the gel. This caused the DNA in the
saline solution or in the damaged wells to not run in proportion to the
correctly filled and intact wells. In order to offset both affects, multiple
trials were performed to clearly distinguish mistakes from true findings.
Figures
Figure
5: Results from Amplified Products Using Gel Electrophoresis.