Revised by: Chelsea Gladney, Emily Henderson and Kara Soronen
We hypothesized that the forward primer, which would attach at the mutation, was more likely to anneal to the DNA sequence if the mutation was located at the 3’ of the DNA sequence. Designing the primer to detect the mutation in the middle of the sequence could lead to false-positive results. The primer can still bind to the surrounding sequence if the mutation is in the middle. To test our hypothesis, multiple PCR tests were performed using primers that were designed to locate and amplify the A455E mutation in the event that it was present in the DNA sample. A control primer was also designed to bind to the DNA if the mutation was absent in the DNA. The A455E mutation is a Class V mutation, resulting in transcripts that are both functional and dysfunctional. Consequently, the number of CFTR channels that reach the apical membrane is reduced. (Kerem et al. 2006). Since the protein is partially functional, the A455E mutation is considered to be a mild mutation of Cystic Fibrosis.
In preparation for designing our primers, PCR tests and gels were run on E. coli bacteria and the lambda virus. The test trials aided in our ability to evaluate the gels and the calculations of the proper annealing temperatures for our designed primers. The first gel resulted in a failed ladder, without which the length of the resulting strands could not be determined. Aside from the failed ladder there was a tear in the gel and no bands were observed. The bacteria did not show most likely due to loading errors. Also, error could have occurred due to improper extraction of bacteria. In the second gel, improvement was seen when a misaligned ladder and wandering bands appeared. These errors can be accounted for due to improper loading techniques and movement of the gel during electrophoresis. Without a straight ladder the resulting band lengths could not be accurately determined. In order to ensure that past errors were not repeated the accuracy of our laboratory techniques were improved upon.
Next, S9 human bronchial epithelial cells were purified through cellular lysis, resulting in the extraction of DNA. The purpose of the extraction was to isolate the DNA that would serve as the DNA being tested for the A455E mutation. The forward primer was designed to bind to the DNA sequence if the mutation was present, while the control primer was identical to the forward primer except for that it would bind to the wild-type sequence. Depending on the presence or the lack of the mutation, either the control primer or the forward primer would anneal. The reverse primer was designed to be 800 base pairs away from the forward and control primer. Thus if the mutation was present in the human bronchial epithelial S9 cells, a band would appear in the gel at 800 base pairs.
Next, PCR was run multiple times using our primers and DNA cocktail. The repetition of PCR was to minimize error and perfect conditions. Positive results were shown with an annealing temperature of 47˚C. This temperature was calculated by using The Rule of Thumb formula. The formula, (4̊C * (#G’s + #C’s in primer) + 2̊C * (#A’s + # T’s)), accounts for all of the base pairs present in the primers. A band was visible around 800 base pairs when compared to the ladder. To further ensure that the band was a positive result, the band was calculated by measuring the distance between the well and the top of the band. The equation 10^(-0.0156x + 4.1678) confirmed that the location of the band was at 831 base pairs. This equation compares distance versus band length and was calculated with a 1 kb molecular weight ladder. In the lane where the control cocktail was loaded, a cloud of the control and reverse primers was present. This is due to non-specific binding and supports that it is not wild-type DNA.
In order to confirm that the band produced was not a false positive result, further testing occurred. First, the primers were diluted, 10 µL of primer to 90 µL of water. The purpose of the dilution was to show that the prior results were actual results, and not a concentrated band of primers. When the gel was run the ladders appeared; however there were no visible bands. This supports that the concentration of the primers was correct. Next, the annealing temperature was lowered to 43 ˚C. While the temperature change produced bands during our trouble shooting experiments, bands were not produced when testing for the A455E mutation. These tests validate that our original testing conditions of non-diluted primers and an annealing temperature of 47˚C were the best conditions for the forward and reverse primers to bind to the mutation. It is not a coincidence that our band appeared where the primers were expected to bind. Further testing could be approached in the future for the most effective location for the primers to bind to the mutation along the DNA strand. We tested our primers with the hypothesis that the most effective binding site would be at the end of the strand, but further tests could determine if another location is more accurate.
During the testing of the designed primers we speculated where Cystic Fibrosis would rank when compared to cancer, heart disease, diabetes and AIDS. To test the public awareness and interest of Cystic Fibrosis we designed and conducted a survey. The survey was administered to our LB 145 biology class who was familiar with Cystic Fibrosis, and three HPS classes. The students were asked to rank in order of importance the five diseases listed. We predicted that cancer would be the most important and detrimental disease to the surveyors because of its prevalence in our society and that Cystic Fibrosis would rank the lowest because it is not as widely publicized.
Cancer was considered the most important to the surveyors, most likely because cancer is currently one of the leading causes of death in the U.S. (Best et al. 2004). The surveyors ranked cancer as being the most detrimental and the disease that should be cured first. A more recent survey was conducted in North Carolina showing that as of 2006 cancer had become the leading cause of death leading us to assume the trend may have grown over the years and further supporting our results (N.C. Public Health et al 2008). As predicted, cancer, AIDS and heart disease were the leading concerns for our survey population with the less publicized diabetes and cystic fibrosis as the least concerning diseases. The most detrimental diseases were cancer, heart disease and AIDS but while the students consider these the worst, if a cure could be found they show that AIDS should be cured over heart disease. This change of opinion over the level of damage to the population and which cure would best benefit them may be a result of the media attention over the AIDS epidemic in Africa (Barnett et al. 1991). If Cystic Fibrosis were more widely publicized then a more accurate public opinion could be taken with most of the population familiar with the five diseases.
The primers we designed can be further applied in science using a multiplex PCR test to detect all mutations that lead to Cystic Fibrosis. This would allow scientists to determine if a patient has one or more of the mutations that leads to this disease. Our contribution to this large scale test would be our designed primers for locating the mutation A455E.
