Written by: Kyle McDonald; Revised by: Lauren Doud; Finalized by: Cara Gonzalez
The E60X CFTR mutation is a single base-pair substitution of G to T at codon 60 of the gene producing a stop codon instead of glutamic acid, resulting in mRNA being prematurely truncated. CFTR is not synthesized, indicating a Class I mutation (Malone et al, 1991). To determine whether a human genomic DNA sample contains the E60X mutation, DNA was purified from IB3 cells using Qiagen Inc. protocols. With designed primers, correct temperatures and PCR cocktail concentrations, the wild-type E60X allele was predicted to amplify and produce a 440bp band in gel electrophoresis. The forward and wild-type reverse primer in PCR annealed to wild-type DNA, which lacks the E60X mutation, because there was no mismatch; thus the wild primer annealed and a 440bp band appeared. When forward and the mutant-type reverse primers were added to wild-type DNA, it prevented amplification of the targeted section of the CFTR gene; the single base-pair mismatch of the mutant-type primer results in no visible bands since it will not anneal to the DNA. This research is significant because while many mutations cause CF, they all differ and the ability to easily diagnose patients for a specific CF mutation is imperative for treatment. In addition, a psychological experiment on 150 Lyman Briggs College students was conducted to examine the influence of positive and negative advertisements on monetary increase of donations to CF research, as continuing research is key for advancing scientific knowledge. The negative advertisement garnered significantly more increased donations, as verified by ANOVA analysis.
Written by: Brianne Falvey; Revised by: Victoria Gonzalez; Finalized by: Kyle McDonald
Written by: Lauren Doud; Revised by: Kyle McDonald; Finalized by: Brianne Falvey
Experiment
The disease Cystic Fibrosis (CF) is caused by a mutation on the autosomal recessive gene on chromosome seven which affects the synthesis of the cystic fibrosis transmembrane conductance regulator (CFTR) (Welsh and Smith, 1995). Previous research has determined that there are over 1000 different mutations known to cause CF, most commonly ÆF508 (Brown et al, 1996). The specific mutation in question for this experiment is the E60X mutation.
The E60X mutation occurs in approximately .05% of CF patients most of who are of Jewish decent (Rowe et al, 2005). This mutation is caused by a substitution of a guanine to a thymine in the 60th amino acid of the CFTR gene, resulting in the production of TAG, a stop codon, instead of GAG, Glutamic acid (Malone et al, 1991). The stop codon causes the polypeptide form of the CFTR protein to be prematurely truncated during translation resulting in the incomplete polypeptide chain being discarded (Rowe et al, 2005).
The E60X mutation is much less common than other CFTR mutations. Despite the small presence of this mutation in the population it is still important to develop a test for the presence of the mutation in patients. In order to create an accurate test primers designs, concentrations of components in the PCR cocktail, durations, and temperatures had to be determined to successfully amplify the target DNA containing the mutation site using the Polymerase Chain Reaction (PCR) method. It was our goal to design a reverse wild type primer, reverse a mutant type primer and a forward primer that would accurately show the presence or absence of the E60X mutation using PCR, allowing for a more accurate diagnosis of CF caused by the E60X CFTR mutation.
In addition to designing a set of primers that would amplify the E60X mutation, we investigated how advertisements influence how much money people are willing to donate to a medical foundation like the Cystic Fibrosis Foundation. This is important to determine the best way to advertise for contributions in order to maximize funding of cystic fibrosis research. Advertisements commonly convey one of two tones: positive or negative. The positive makes the viewer feel optimistic about the difference they would make to the foundation due to their contributions. The negative portrays a sad message that invokes powerful guilty or remorseful response from the viewer, making them feel obligated to donate a greater amount of money to the foundation (Dillard and Peck, 2000). For our experiment we had three different advertisements; one positive, one negative, and one control. Each advertisement was shown to three different sections of the Lyman Briggs Calculus I classes of about 50 students each. Prior to seeing the question the study groups were asked to write down how much money they would donate ranging from $0-20 in four-dollar increments. This served as a pre-measure to compare how the advertisement actually effects how much money each person is willing to donate. After this, the advertisement was shown and the group was once again asked to write down how much money they would be willing to donate.
Original Predictions
Using PCR and DNA taken from human cells, the E60X allele was amplified, and then interpreted by running gel electrophoresis to determine whether the mutation exists. A successful test results in the target DNA strand with a length of 440 base pairs. This length was determined because the forward and reverse primers are designed to be 402 base pairs away and are 20 and 18 base pairs long respectively. The primers were designed at these specific lengths so the annealing temperatures between the forward and reverse primer would vary by only one degree Celsius (Kibbe, 2007). Our hypothesis is that by using the primers designed, a band of 440 base pairs in length will appear when wild type DNA is reacted with wild type primers and not when reacted with the mutant type primer, allowing for an accurate diagnosis of the presence or absence of the E60X mutation. The E60X mutation will be shown absent when the forward primer and the wild type reverse primer are reacted in PCR with the DNA of a person that lacks the E60X mutation, a band of 440 base pairs in length is present in the gel electrophoresis. In addition the forward primer and the mutant type reverse primer are reacted with the DNA of a person that lacks the E60X mutation, no visible bands will appear in area where a 440 base pair segment of DNA would occur compared to the DNA ladder. This is because mutant primer was design to only anneal with DNA that has the E60X mutation, otherwise the annealing and extension will continue normally, making a much longer band of DNA.
If no band is present when the wild type primer and wild type DNA is reacted together or if a band is present when the mutant type primer and wild type DNA is reacted it would show an error in our hypothesis. Possible errors include; inaccurate primer design, incorrect annealing temperatures, incorrect salt concentration or pH in the buffer solution for the Taq polymerase to properly function, and incorrect lengths of cycles. When specific types of advertisements were analyzed to determine their influence over the amount of money people are willing to donate to a medical cause, we predicted that the negative advertisement would inspire the greatest change in the amount of money willing to be donated. This is because the negative advertisement is designed to make the viewer feel a sense of remorse and guilt that they are almost required to donate more to help those in need (Dillard and Peck, 2000).
Findings
From the data collected concerning the type of advertisements and the change in the amount of money people were willing to donate to a cystic fibrosis research we were able to determine that the negative advertisement is most affective at promoting higher donation rates. When comparing the pre-measure and the post-measure donation amounts for the negative advertisement 51.35% of individuals did not change the amount of money they would donate, 29.73% of individuals increased their donation by $4, and 18.92% of individuals increased their donation by $8. For the group that viewed the positive advertisement 74.47% did not change the amount of money they would be willing to donate, 19.15% of individuals increased their donation by $4, and 4.26% of individuals increased their donation by $8. For the control advertisement 86.54% of individuals did not change their donation amount, 11.54% changed their donation amount by $4, and none change their donation amount by $8. The negative advertisement had the lowest percentage of individuals that did not change the amount that they would be willing to donate and it also had the highest percentage of individuals that would donate $4 and $8 more after viewing the advertisement. An ANOVA test was performed to determine the significance of this data and results showed a significance between the negative and positive and the negative and control advertisements, but no significance was found between the control and positive advertisements. The overall p-value was <0.01 supporting that the results found were significant. These results indicate that the negative advertisement elicits a more powerful response which encouraged a large increase in donations when compared to the positive and control advertisement. The positive was not significantly different from the control, indicating that this advertisement scheme is not as affective at garnering donations from the public for medical research. Therefore when research groups attempt to gain funds from the public an advertisement that produces a remorseful or guilty feeling is more affective.
Future Directions
The diagnostic assay developed in this experiment could have been improved by providing further confirmation of the identity of our expected target DNA. Many tests could have provided this confirmation. One test would be to find DNA with the E60X mutation and run PCR with the same primers. In this case, the E60X-containing DNA with the mutant type reverse primer should produce a band at 440 base pairs in length and the E60X-containing DNA with the wild type reverse primer should produce no band. If these results were obtained this would provide further support that our primers annealed to the correct location of the DNA. PCR with restriction enzymes would also provide support. A restriction enzyme chosen that would cut at the location of the mutation could be used to cut our target DNA. By determining the expected lengths of the fragments that should be produced, a gel could be run, and if it showed bands at these lengths, it would further confirm the identity of our band. Another method for strengthening our diagnostic assay would be to design a set of Yaku primers similar to the traditional primers to see if they produced clearer bands.
To further improve and expand the advertisement research, several factors could be changed. Because subjects were only taken from a Lyman Briggs calculus class, the sample is not very diverse. If a broader range of ages, classes, and professions were used, regional bias could be reduced. For example, college students are tight with their money and are not as financial aware as the older population and may choose dollar amounts differently because of that. If the sample size was increased as well, there could be a better representation of all potential donors. In addition, a larger sample size would allow for smaller dollar increments, such as $1 increments. More available data would allow for a more accurate statistical analysis.
Written by: Lauren Doud; Revised by: Kyle McDonald; Finalized by: Brianne Falvey
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