Diagnosis of G542X mutation in bronchial epithelial cells of cystic fibrosis patients using PCR
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Marissa Albright, Amanda Bartenbaker, Kristina Kunze, Melissa Levine Abstract
Cystic fibrosis (CF) is a disease caused by recessive genetic mutations in the cystic fibrosis conductance regulator, CFTR (Welsh and Smith 1995). Research was conducted to determine if G542X, a mutation known to cause CF, was present in a DNA sequence. This mutation is the second most common among those with the disease (Hopkin 1998). DNA from human bronchial epithelial cells was purified and run using Polymerase Chain reaction (PCR) with specific primers designed for allele specific amplification. The primers were designed to build toward each other with one primer attaching to the mutation and another attaching on the complimentary strand 600 base pairs away. The results were viewed using 0.8% agarose gel with UV illumination. Our hypothesis was that the best detection of G542X would come from the mutation being on the 3' end of the primer. Our results did directly not support or refute our hypothesis. In order to see the effects that preimplantation genetic diagnosis (PGD) would have on the frequency of CF in a population, a survey was conducted to determine under what circumstances people would undergo PGD. The results were combined with existing statistics to create a model of the change in frequency of the disease over five generations. Our hypothesis was that a majority of the people would undergo PGD and that the frequency of CF would drastically decrease over time. The results showed a significant logarithmic decrease in cystic fibrosis over time. Figure
Surveys were given to a group of individuals asking under what circumstances they would undergo Pre-Implantation Genetic Diagnosis. The results of the individuals' responses were used to determine the affect it would have on the frequency of cystic fibrosis over time. The graph shows genotype frequencies over time for the first five generations. The points above were plotted on a graph and a line of best fit was used. Subsequent generations can be calculated using the equations shown above. Discussion Cystic fibrosis can be caused by many different mutations of the genes which create Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The most common of these mutations is ΔF508, which causes a deletion of a phenylalanine amino acid within CFTR (Wright et al, 2009). The mutation studied in this experiment was G542X, which is the second most common mutation worldwide, and has an increased frequency in Mexican, Spanish and Jewish populations (Hopkin 1998). The objective of this research was to design a protocol that could test for the G542X mutation. This was done as a preliminary to a complex assay that will be designed in the future which will have the ability to test for multiple cystic fibrosis mutations at once. Polymerase Chain Reaction and Primers The Polymerase Chain Reaction method was utilized for allele specific amplification. Primers were designed with the mutation base located on the 3' end of the primer with the idea that the Taq polymerase would be less likely to bind to a dangling unattached base; whereas if the mutation were in the middle of the primer, the primer could make a ‘U' shape around that base and attach the rest of the way (figure 1) (Coleman and Tsongalis 2006). We hypothesized that by placing the mutation position on the 3' end of the primer, the Taq polymerase would be less likely to bind onto uncomplimentary bases, thus reducing the likelihood of false positive results. The products of PCR were analyzed using 0.8% aragose gel electrophoresis and ultraviolet illumination. A band size of 600 base pairs was expected in Lane 1 if the DNA did not contain the mutation or Lane 2 if the mutation was present. No bands were expected in the C1 or C2 lanes because they did not contain any DNA. Eight trials were run, utilizing different primer annealing temperatures and phase times. No 600 base pair bands appeared in the gels of any of the trials. These results do not refute or support our hypothesis. Were a 600 base pair band to show up in either Lane 1 or 2, we could assume that the primers had annealed and the hypothesis would have been supported. If bands showed up in both Lanes 1 and 2 the hypothesis would have been refuted, because it would have meant that the Taq polymerase had bound to and amplified the DNA regardless of the base pair mismatch. We do not believe that the absence of these results is ample support to refute our hypothesis. The primer bands present at the bottom of the gels for Trials 1, 3, and 5 demonstrate that the primers did not attach at all. Due to the number of variables that affect PCR, we simply did not have enough time to find the precise phase times and annealing temperature required for our primers. However, we did collect enough information to make other conclusions. Conclusions based on PCR with Lambda DNA A trial run of PCR and agarose gel analyzation was completed with Lambda genomic DNA in order to test the protocols involved. The resulting gel contained a band about 500 base pairs long. This outcome coincided with the expected band of approximately 500 base pairs , which was the distance between the primers used. This success demonstrated that the volumes and components for the PCR cocktail were correct. Since these same amounts were used for our trials for the G542X mutation, cocktail composition can be ruled out as a possible factor for the failure of those trials. DNA Purification A DNA purification of cultured IB-3 human bronchial epithelial cells from CF patients using the kit from Qiagen Inc was also conducted. We verified the purification by two different techniques; spectrometric analyzation and gel electrophoresis. We were under the impression that the resulting number from spectrometry at a wavelength of 260/280 should yield a ratio around 2. We concluded that our DNA was pure because our number (3.0285) was close to 2. Upon research done before trials 7 and 8, we concluded that this test did not verify the purity of our DNA. Purified DNA should yield a ratio between 1.8 and 2.0. This number is the ratio of the absorbance at 260nm (the wavelength DNA absorbs best) versus absorbance at 280nm(the wavelength proteins absorb best) (Heaton, 1999). In addition, when we used gel electrophoresis to further verify the DNA was purified we concluded that because the smear in the gel ended with a clear band it was indicative of a pure sample. Again, later research indicated that a smear above the band indicates degradation of the DNA and can interfere with PCR (Nollet, 2004). Therefore, the template DNA used for Trials 1-6 may have been present in pieces instead of a whole strand and prevented proper binding and extension of the primers. To correct for this, we used another group's purified DNA, which did yield a clear band when run on a gel, for Trials 7 and 8. Gels As addressed before, the Trial 1 gel yielded primer bands in lanes 1, 2, and C2. This indicates that our primers did not anneal to the template DNA. The two main reasons we hypothesized that they did not anneal were that either the phase times did not give enough time for the primers to find their complimentary strand and anneal or that the annealing temperature was wrong. We decided to change one variable at a time so that we could proceed in a methodical manner. First, we increased the phase times to 45s. Trial 2 produced a gel with a successful ladder, but nothing else. Since the increased phase times did not produce the expected results, we reduced the phase times back to the original 20s and decreased the temperature for increased specificity for Trial 3. This was our most successful gel in that we obtained a smear in Lane 1. We decided that perhaps the smear was due to too much DNA, so we ran a second gel with decreased amounts of products in the wells. This gel yielded a ladder but no other marks, which allowed us to conclude that too much DNA was not the issue. For Trial 4 we returned to increased phase times and performed a 1:10 dilution of the primers to see if too much primer was an issue. This gel also had a successful ladder, but no bands whatsoever. Due to the fact that we had obtained a smear in Trial 3, we decided to not dilute the primers in the consequent trials. Trial 5 was identical to Trial 4 save that the primers were not diluted. Again, a ladder appeared and primer bands were present. This indicatesdthat the primers were not annealing. We raised the annealing temperature two degrees for Trial 6 to see if that temperature would be the key one. It was not; the gel had a faint ladder and two primer bands. At this point, we did some research and found that our DNA might not have been purified. We did not have time to purify more DNA and our resources were dwindling, so we borrowed DNA from another group who did attain a band in order to rule out un-purified DNA. We also found an equation that stated phase times should be 1 minute for every 1000 base pairs the expected band will be (Sambrook and Russell 2006). This came to 36 second phase times. We had also read and found that a final extension is sometimes included in PCR and should be between 5 and 10 minutes long (Bartlett and Stirling 2003). For Trials 7 and 8 we used a shorter hot start, 36 second phase times, and added an extension. For Trial 7 we used an annealing temperature of 50 °C because we had obtained a smear at that temperature in Trial 3. Trial 8 had an annealing temperature of 54°C because it was in between other temperatures that had been used in previous trials. Both of the gels yielded no ladders and no bands. This indicated that there was something wrong with the gel. Since both were run at the same time with the same voltage source, a problem may have arisen. More likely is that Ethidium Bromide was not added properly when the gel was being made. The dye in the product indicated that the product had traveled a sufficient length down the gel when it was analyzed. Two more gels were run using Trial 7 and 8 products and also yielded no results. The ladders were not defined and no bands were present. Future Directions We can conclude that the combinations of phase times and annealing temperatures that we chose do not work for our primers, assuming that the DNA we used was indeed purified. If this research were to be continued we would take into account the degregation of DNA, and use freshly purified DNA for each trial. More trials would be run with a plethora of different annealing temperatures in order to find the perfect one. We believe that the 36s phase times are correct because of the equation that was found. Perhaps annealing temperatures below 50°C could be used to increase specificity (Sambrook and Russell 2006). |
