Abstract
Written by: Kayla Stewart
Revised by: Megan Schalau and Layne Weatherford
The purpose of our study is to design primers for a PCR assay that will detect the single base pair mutation 1078 del T, on the CFTR gene of patient DNA templates. The experimental design consisted of four PCR assays: two mutant (MT) DNA reaction cocktails and two wild-type (WT) DNA cocktails (Wittwer et al., 1993). This was analyzed by gel electrophoresis under UV light to find the specific band lengths obtained. A Qiagen Inc. generation capture column kit was used to isolate and purify the genomic DNA from IB3 (MT) and S9 (WT) human bronchial epithelial cells.
We hypothesized properly designed primers would result in DNA amplification. Based on research done by I.
Dorval and others, we hypothesized PCR would result in mutant and reverse primers annealing to MT DNA at 50° C,
giving a gel band at 3,603 base pairs (bp). Wild-type and reverse primers were expected to anneal to WT DNA at
50° C, resulting in a 3,603 bp gel band. The results obtained were skewed because of incorrectly designed primers
resulting in nonspecific binding. Further CF studies could result in one PCR assay used to screen populations for
multiple CFTR mutations previously studied (Dorval et al., 1994; Moullier et al., 1993). A sociological survey was
also administered to 200 people asking them what their views were on the topic of abortion, cystic fibrosis screening,
and the economy. Based on research by D.C. Wertz and associates, the hypothesis was that the majority of people
would not consider abortion as an option for fetuses screened positive for CF. The results supported this.
Discussion
Polymerase chain reaction is a popular technique used for DNA amplification (Wittwer et al., 1993). Once the procedure replicates the DNA, the products of PCR can be analyzed using agarose gel electrophoresis (John et al., 2009). For our investigation we designed primers for a PCR test to screen for the mutation 1078 del T in the CFTR gene that causes a severe form of cystic fibrosis. Primer 1 (p1) was intended to anneal during PCR for a reaction cocktail containing a mutant DNA template. For wild-type DNA, primer 3 (p3) was intended to anneal in the case of mutation absence. We hypothesize that properly designed primers for the CF mutation 1078 del T will anneal at 50°C during PCR resulting in a DNA amplification product 3,603 base pairs in length. Unfortunately, both the mutant and wild-type forward primers were designed incorrectly. They were designed to anneal to the anti-sense strand, the same strand the reverse primer was designed to anneal. As a result of this error, the forward and reverse primers extended in the same direction and, therefore, did not amplify the specified segment of DNA. Ultimately, due to incorrect primers our test could not accurately discriminate between wild-type DNA and DNA containing the 1078 del T mutation. Additionally, DNA containing the mutation 1078 del T was unattainable, so IB3 cells were used instead. We hypothesize that if the forward primers had been designed to complement the sense strand rather than the anti-sense strand, and DNA containing our mutation researched, that the assay would have properly detected the presence of the 1078 del T mutation.
Due to the single base pair mismatch of the 1078 del T mutation, conventional PCR annealing temperatures must be near exact for proper DNA replication to occur. A study performed by Carl T. Wittwer and others showed that rapid cycle allele-specific amplification allowed for single base-pair discrimination at various annealing temperatures, so there can be more room for error without sacrificing desired results (Wittwer et al., 1993). Their studies comparing rapid cycle allele-specific amplification and PCR had shown conventional PCR requires exact annealing temperatures for primers to properly discriminate a single base-pair mismatch while rapid cycling was effective at annealing temperatures of 50°C or lower (Wittwer et al., 1993). According to Wittwer, rapid cycle amplification is quicker and more efficient, but this technique was not available. Instead, a thermocycler was used to perform conventional PCR. As a result, the annealing temperatures must be exactly right for the primers to anneal and allow for amplification.
In Rapid Detection of 1078 del T Mutation by PCR-Mediated Site-Directed Mutagenesis: Detection of Cystic Fibrosis Carriers in a Celtic Population, a study conducted by Ian Dorval and his associates on the mutation 1078 del T, PCR was successfully used to detect the mutation. Furthermore, a specific restriction enzyme was used to confirm the presence of the desired bands in the gel. The restriction enzymes were designed in such a way that they cut the wild-type DNA at one recognition site, but two recognition sites were present in the mutant DNA. The electrophoresis resulted in two bands for the wild type allele and three bands for the mutant allele, indicating that the primers properly discriminated between wild-type and mutant DNA and that the desired bands were amplified. The designed primers and restriction enzyme from this particular study created a way to detect carriers of the mutation as well (Dorval et al., 1994). The efficiency of a PCR assay detecting the presence of 1078 del T is crucial because it has been compared in severity to the most common mutation causing cystic fibrosis: ∆F508, with mortality rates only slightly less than the ∆F508 mutation (Moullier et al., 1993). Failure to design correct primers inhibited our ability to produce the desired bands in the gel and confirm them with restriction enzymes.
Unfortunately, none of the five gels run throughout our research produced the desired 3,603 base-pair length band. Errors in PCR were due to incorrectly designed primers which failed to amplify the intended strand of DNA and, therefore, could not indicate whether DNA had the mutation 1078 del T or not. We observed smearing at an annealing temperature of 50°C that may be due to non-specific binding or too much DNA in the PCR cocktail. The brightest bands using the wild-type forward primer were found in lanes 1 and 2 and were 2,000 base-pairs in length. The brightest bands using the mutant forward primer were found in lanes 3 and 4 and were 3,000 base-pairs in length. For this reason, we believe if correct primers are used, an annealing temperature of 50°C would allow for proper annealing and amplification. The reason no product was visible in 3 of the 5 gels could be due to not enough DNA in the reaction cocktail, but is most likely due to the incorrectly designed primers being unable to anneal and allow for amplification. With both the forward and reverse primers extending in the same direction, the segment between the primers was not amplified. For the gel in which no PCR product or 1 kb ladder was observed, the gel electrophoresis was run for 45 minutes at 131 Volts. The gel was run too long, so any product that may have been present along with the 1 kb ladder traveled past the end of the gel.
A multi-dimensional PCR test could be designed to screen patients for multiple CFTR mutations using a collaboration of adequately designed primers. If the annealing temperatures for the several primers detecting the various mutations can be made similar and the resulting amplified DNA fragments were unique in their base-pair band lengths, then one assay could be used to detect multiple cystic fibrosis mutations. If the multi-dimensional PCR screening is efficient in testing CF patients it could potentially be used for prenatal CF detection. Since the discovery of fetal DNA circulation in maternal plasma and serum, non-invasive procedures have been performed to obtain prenatal mutation diagnosis without any harm to the fetus (Gonzalez et al., 2002). If screening is able to detect multiple mutations, the test will be much less expensive than using several tests to check for various different mutations. Also, if diseases like cystic fibrosis can be screened for and identified early, patients and their families will be better prepared to cope with the disease and receive the care necessary to provide the best quality of life possible for the individual diagnosed with CF.
As an extension of our investigation we conducted a sociological survey in regard to the ethics and economics of
aborting a fetus with cystic fibrosis. We surveyed 200 people, the majority of whom were Michigan State University
students. A study conducted by D.C. Wertz and associates surveyed 271 parents of children with CF and revealed that
20% would choose to abort a fetus with CF (Wertz et al., 1991). A subsequent study performed by the same
researchers produced very similar results. Of the 227 couples surveyed, 20% said they would abort a fetus if prenatal
screening revealed the child would have CF (Wertz et al., 1992). Also, the U.S. economy is in the middle of a
recession, and people all over the country are affected by the 8.5% unemployment rate and the increasing health care
costs (U.S. Bureau of Labor Statistics). According to research performed by C. Krauth, the direct cost of care for
someone with cystic fibrosis is between 6,200 and 16,300 U.S. dollars a year (Krauth et al., 2003). The goal of our
study was to determine whether the economic burden of having a child with cystic fibrosis would affect the decision
whether or not to abort the fetus. Because previous studies concerning abortion and CF showed that only 20% of
parents of CF patients would terminate a fetus with CF, we hypothesized that after giving people background on CF
the majority of people would still not choose to abort a fetus screened positive for CF regardless of economic or
financial conditions. Of the 200 people surveyed, 69% initially said they would not consider aborting a fetus screened
positive for CF; 40% (80 people) were female and 29% (58 people) were male. Of these 58 men, 8 (14%) indicated
that the economic situation of a family would affect whether the fetus was aborted or not. 24 of the 80 women (30%)
agreed that the financial status of a family would factor into the decision of whether or not to abort the fetus. Overall,
23% of the people surveyed who initially said they would not consider pregnancy termination as an option if their child
was screened positive for CF later indicated that economics would affect their decision. Therefore, a total of 106
(53%) of the people surveyed would not abort a fetus with CF regardless of economic conditions. These results
support our hypothesis that the majority (53%) of people would choose not to abort a fetus with CF regardless of
economic conditions.
Written by: Eric Tye
The PCR assay to detect the mutation 1078 del T failed because both forward primers and the reverse primerwere designed to anneal to the anti-sense strand, leading to extension in the same direction and no amplification of the desired band. In order to redesign the forward primers correctly, they would have to be designed to anneal to the sense strand. In other words, they would complement the sense strand and extend toward the reverse primer allowing for the desired amplification of DNA. The single base-pair mismatch would be located at the 3’ end of the mutant primer and, therefore, would properly discriminate between DNA containing the 1078 del T mutation and wild-type DNA. Also, if time permitted the correct amplified band of DNA 3,603 base-pairs in length would be sequenced in order to confirm that it was indeed the desired band. Sequencing would reveal each base-pair present in the amplified band of DNA in 5’ to 3’ order. This sequence could be compared to the expected sequence of base pairs between the forward and reverse primers that were expected to amplify. If they are the same, then the primers annealed at the desired locations and the correct band was amplified. We could also use restriction enzymes to confirm the correct band was amplified. We would use a restriction enzyme that has a recognition site present somewhere in the amplified band sequence. Knowing the recognition site, we could determine band lengths that should result from the cutting of the restriction enzyme. If the expected band lengths correspond to the bands observed in the gel, then we would know the correct band of DNA was amplified.
The primers may have annealed to the wrong locations on the DNA, resulting in non-specific binding. The bottom most bands are likely primers.