Gene Expression of band amplification in S9 cell line of CF patient for A455E mutation employing Allele Specific PCR

By: Elizabeth Kim, Rebecca Lust, Kevin Panasiewicz, and Lucas Zavadil

The Green Berets

Abstract

The A455E mutation involves a change in the 1496^th nucleotide, changing the codon alanine to glutamic acid at the 455^th amino acid on Exon 9 in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein (Strasburg et al., 1997). This mutation hinders the passage of chloride and sodium ions through the CFTR protein, resulting in more mild symptoms compared to severe mutations (O’Sullivan and Freedman, 2009). Purified DNA from an S9 cell line CF patient was observed using allele-specific PCR to identify its genotype as homozygous wildtype, heterozygous, or homozygous for the A455E mutation. The Yaku-Bonczyk method, described later on, was used to design the primers. We hypothesized that a single base pair mismatch could be used to determine the genotype of the purified DNA and ultimately, the presence or absence of the A455E mutation. After observing the gels, a 542 bp band expressing a homozygous-wildtype genotype was found. This experimental approach can identify the presence of other allele-defined cystic fibrosis mutations in patients and prospective parents. Also, a study was performed to observe a possible correlation between university majors and education received about lethal genetic diseases such as CF. By distributing a survey to an ISS 320 sociology class, it was hypothesized that biological science majors were more knowledgeable about cystic fibrosis and supportive of genetic testing and research. 36.47% of biological science majors answered questions about CF correctly, only 2.44% higher than other non-science majors. The average response for all majors was 3.612. Data shows a student’s major is not correlated with his or her knowledge of a genetic disease and willingness to support genetic testing and research.

Discussion

Experiment Summary

Cystic fibrosis, an autosomal recessive disease, is due to genetic mutations on chromosome 7 which causes alterations in the performance or presence of a single protein, the Cystic Fibrosis Transmembrane Conductance Regulator or CFTR gene, which functions to transport chloride ions through the apical surface of epithelial cells (O’Sullivan and Freedman, 2009). Thusly, the symptoms of cystic fibrosis, most notably respiratory failure in the severest of cases such as with the prominent ΔF508 mutation, are directly related to the excessive mucosal upsurge and subsequent bacterial infection and inflammation resulting from the malfunctioning of that particular protein (Welsh and Smith, 1995). There are numerous mutations of varying severity identified for CF, one of such and the focus of this study is the A455E mutation, which causes a nucleotide base change from C to A at 1496 and a codon change at the 455^th amino acid within the Exon 9 region of the CFTR genomic DNA (Strasburg et al., 1997). While PCR has been proven to be an effective means of diagnosing the presence of genetic disorders (O’Leary et al, 1997), the question being addressed is if a PCR test can be designed to recognize this particular mutation. We hypothesize that by designing specific forward and reverse primers designated by the Yaku-Bonczyk method (Yaku et al, 2008) and employing the PCR protocol for DNA synthesis, followed by agarose gel electrophoresis of the DNA, we will be able to obtain an allele-specific amplified band displaying whether the tested DNA is homozygous for the A455E mutation, heterozygous for the A455E mutation, or whether the DNA is homozygous wild-type and therefore does not express the mutation.

Along with designing primers to test for the presence of a specific mutation a survey was conducted to determine the correlation between the awareness of cystic fibrosis and risk management. For the sociological experiment it was hypothesized that, through applying a broad-pool survey linking student majors and their knowledge of cystic fibrosis, there will be an increased public awareness of CF among those majoring in biological sciences. Discussing risk assessment, management and communication with Toby Ten Eyck, the professor of ISS 320 Risk & Society at Michigan State University and author of an article relating public awareness and biases towards genetic testing with regards to the media, will hopefully lead to the conclusion that those who hold a better understanding of rare diseases will be more likely to support funding for research pertaining to the genetic identification and cure of those diseases (Ten Eyck, 2005).

Original Predictions

The actual primers for this study are 5'-GAAAGAGGACAGTTGTTTGA-3' for the forward primer corresponding to the mutated anti-sense DNA strand, 5'-GAAAGAGGACAGTTGTTTGC-3' for the forward primer binding to the anti-sense wild-type DNA, and 5'-TCCTCAAAAAACTATCCCAC-3' for the reverse primer complementary to the sense strand 502bp away on the genomic DNA. Each primer is, most efficiently, 20bp in length with annealing temperatures of 48˚C, 50˚C, and 48˚C correspondingly, and will be binding to a DNA template lysed and purified from preexisting Cystic Fibrosis cells (Haenisch et al., 2009). Through the use of these primers in conjunction with a negative control, either water or a PCR cocktail without a DNA template or primers, in gel electrophoresis, a gel band of 542bp is expected to be amplified for the annealing of the forward mutant primer to the reverse if the mutation is present or with the forward wild-type primer if it is not. With this result, the primers and methods used to obtain that band will represent an accurate application for detecting the A455E mutation in a given DNA sample based on the alleles. This would validate our hypothesis and, in turn, confirm whether a PCR procedure for allele-specific detection of this mutation can be identified, utilized and replicated. For the sociological experiment it was expected that students majoring in the biological sciences would have a greater knowledge of CF and express a greater interest in CF risk management strategies.

Results and Findings

Multiple experiment PCR trials were run in order to determine the optimal PCR conditions. There were adjustments in annealing temperature, primer concentrations, buffer type and addition of extra magnesium. Through the various trials it was determined that the optimal annealing temperature for these primers was 46˚C when setting the thermo-cycler to run for 30 cycles. The correct band of 542 base pairs correctly appeared when using 1 µL of primers with the LB buffer and no extra magnesium. The band also appeared slightly brighter with the addition of 1 µL of magnesium chloride. However, using the magnesium chloride also resulted in a gel with greater amounts of smearing due to non-specific binding. The band appeared in the lanes corresponding to the use of the wild-type primer only. This means that the DNA template used to run the PCR test did not contain the mutation that was being tested for. Acquiring bands of the expected length supported the hypothesis of using Yaku primers as a viable method of diagnosing the presence of the A455E mutation. However, these primers lead to an overabundance of smearing most likely due to non-specific binding. This would then lead us to refute our hypothesis because this test is not an accurate means for determining the presence of the A455E mutation.

The results of the sociological survey ended up being nothing like what was predicted. In the first five questions students majoring in biological sciences only answered 2.44% higher than all of the other students surveyed. Every single major possessed a small amount of knowledge based around CF represented by 94.68% of the students surveyed correctly answering the first question. For the remainder of the first five questions a unique major represented the group with the highest percentage correct. Overall knowledge of CF has no correlation with ones major. For questions 6 through 10, the average responses among students of any major was either indifferent or somewhat supportive of risk management strategies, such as genetic testing at various stages and research funding, for rare diseases such as cystic fibrosis. A major in the biological sciences was not directly correlated to support of these avenues, nor was it indicative of a greater awareness of the disease, a factor that was predicted to positively influence the support of these strategies. This result represents an idealistic view among these students. Given that the average student received a 1 on the awareness scale, the students are employing subjective reasoning as opposed to objective, generalizing and therefore diminishing the survey’s goal of obtaining their views on these methods for cystic fibrosis in particular. Although there were much fewer “strongly agree” responses, the class’s tendency for support of preventative measures may be influenced to their opposition towards recent nationalized healthcare debates, expressing their dissatisfaction by agreeing with statements that, in a manner, contest the current administration’s goals for the health system.

Future Direction

A possible means for experimental improvement could be designing a second set of primers to complement the ΔF508 cystic fibrosis mutation’s phenylalanine amino acid deletion, which was present in this study’s CF S9 cell line genome and, generally, would be the most likely to yield results in an unknown DNA sequence as it accounts for the majority of cystic fibrosis cases (O’Sullivan and Freedman, 2009). Also regarding primer design, minimizing the A/T and G/C complements between the sense and anti-sense strands designated by the forward and reverse primers would reduce the probability of the primers binding to one another, resulting in the primer dimers present in various PCR trials conducted for this study (Haenisch et al., 2009). To increase the clarity of the band, magnesium and other salt concentrations could be increased in various increments, as one additional microliter of magnesium chloride significantly improved the brightness in the 5^th PCR trial conducted (Figure 4).

To improve the accuracy of the sociological survey, additional major-wide, university-required lectures could be included to provide for a larger survey pool. Also, the questions determining the participants’ willingness to support risk management strategies would produce more personal responses by not only having the student state their position on research funding and genetic testing, but whether their position would be altered if they themselves were afflicted with cystic fibrosis. Finally, given that the majority of university students are not yet parents, presenting this survey to older participant who are parents would provide for broader generalization and would reduce the subjective, politically-influenced opinions of a younger survey pool.

Lanes: 1 2 3 4 5 6 7

510, 540

542

1010

400

Figure 3. Electrophoresis Gel from PCR Trial 4, October 21, 2009.

This figure represents the first successful assay of an electrophoresis gel after performing PCR with an annealing temperature of 46 ˚C. This PCR test involved two PCR cocktails where the amount of Yaku-Bonczyk primers was changed. However, both cocktails totaled to an amount of 50 µL by changing the amount of water in the cocktails. Lane 1 contained the 1-kb DNA ladder which was used as a size marker. Lane 2 contained the PCR cocktail with 2 µL of the forward mutant primer, and lane 3 contained the cocktail with 2 µL of the forward wild-type primer. Lane 4 consisted of the PCR cocktail with 1 µL of the forward mutant primer while lane 5 contained 1 µL of forward wild-type primer. Lane 6 was the negative control where no DNA template was added into the cocktail. The failure for a band to appear shows that the DNA template and primers were not contaminated. There was a very faint band in lane 5 510 base pairs long. The faintness of the band could have been resulted from the Ethidium Bromide not being well mixed into the agarose mixture or not enough cycles being run during PCR, leading to less amplification of DNA.