Vincent Cracolici
Laura Gnyp
Stephanie Norris
Kathleen Szczegielniak
G542X is a single base pair genetic mutation, which triggers a stop signal to prematurely stop the formation of CFTR, and is known to cause Cystic Fibrosis (Loirat et al, 1998). The goal of this investigation is to devise an assay that will successfully determine if a certain person carries the G542X mutation. Utilizing PCR, we were able to identify a region of DNA from cystic fibrosis patients where the mutation resides and analyze it for the potential presence of the G542X mutation. We hypothesize that primers that compliment the mutation will anneal to DNA of a person that carries the mutation because they share a nucleotide base pattern, and primers that compliment a normal DNA strand will not anneal to a person carrying the G542X mutation because the nucleotides will not match. The primers anneal to the DNA at 52ºC. We predict that we can diagnose whether or not a subject carries the G542X mutation based on which primers anneal to their DNA and if the PCR showed a band at 902 base pairs in gel electrophoresis because the primers were designed 902 base pairs apart. (Wright et al, 2009). The results obtained were ambiguous. All eight gels showed nonspecific binding. Further troubling shooting needs to be performed such as altering annealing temperatures or primers to obtain the predicted bands. This research attributes to the rapidly growing field of genetic testing and deals with a mutation that is common in specific populations. Gaining insight could help those with the disease to get the best treatment possible.
By designing specific primers that have an affinity for either wild type DNA or mutant G542X DNA, we have created a test that would screen a patient for this CF causing mutation and give them a definitive diagnosis (Loirat et al, 1998). This test, performed via PCR, used specific primers that only anneal to DNA when it is either wild type or mutated, and then these annealing results were made visible through gel electrophoresis. By comparison to a standard DNA ladder, if bands of length 902 base pairs are visible in the gel, then we can conclude whether or not the person carries the mutation. We hypothesized that primers that compliment the mutation will anneal to DNA of a person that carries the mutation because they share a nucleotide base pattern, and primers that compliment a normal DNA strand will not anneal to a person carrying the G542X mutation because the nucleotides will not match.
Originally, the G542X mutation DNA complimenting the mutated nucleotides TGA was predicted to anneal to primer 1 at 54˚C. Likewise, the wild-type DNA that compliments the wild-type nucleotides GGA was also predicted to anneal to primer 3 at 55˚C, and not to primer 1. All DNA was predicted to anneal to primer 2 at 55˚C because this DNA sequence is 902 base pairs away, and both wild type and mutant DNA should be identical at this locus (Wright et al, 2009). The annealing temperature for PCR should be at 54˚C for all three primers. The original phases predicted for PCR, which was run 35 cycles, were: initial denaturing: 5 minutes, denaturing: 30 seconds, annealing: 30 seconds, extension: 60 seconds, final extension: 7 minutes. In the reaction cocktail, the initial amount of purified genomic DNA was 0.75 uL and at first the 1.25 uL of the primers were diluted 10 times. Overall, the annealing temperature for all primers illustrated the best results on the electrophoresis gel at 52˚C. The ending PCR time was: initial denaturing: 5 minutes, denaturing: 30 seconds, annealing: 30 seconds, extension: 45 seconds, final extension: 7 minutes (Wettwer, 1993). The end amount of purified genomic DNA used in the reaction cocktail was found to be 1.5 uL. Also, the primers were sharper in the gel when not diluted and 1.25uL of each was used.
The results of the first PCR trials run did not showcase any sort of amplification (Figure 2). The first trial shows primers at the bottom of the gel, indicating that the primers did not anneal. Because the primers have a small amount of nucleotides they travel through the matrix of the gel faster and further than strands of DNA with many base pairs (Wright et al, 2009). Since the concentration of our purified genomic DNA was low (4.7615 ug/mL), we concluded that there was only a small amount of DNA in the reaction cocktail, not enough for the primers to anneal and be expressed on the gel. Therefore, in the second trial, different volumes of DNA were added to the reaction cocktails: 1uL, 1.25uL, and 1.5uL. The electrophoresis gel showed primers at the bottom and no DNA left in the well, concluding the DNA may not be the issue because the DNA was able to leave the well; however, it was not definitive because the primers did not anneal. Since there was no streaking on the gel, we presumed that the annealing temperature was too high; we lowered it to 51˚C for our third PCR trial, keeping the DNA amount at 1.5uL. The results for the third PCR trial illustrated primers at the bottom of the gel and streaking, indicating that the annealing temperature was not accurate and the primers were not able to anneal correctly. Thus, the annealing temperature was kept at 51˚C while we used both 1.25uL 10X diluted primers and 1.25uL non-diluted primers. The gel electrophoresis for the fourth trial showed the primers at the bottom of the gel with the non-diluted primers being sharper than the diluted primers, and streaking. The annealing temperature was lowered to 49˚C for the fifth trial. The electrophoresis gel showed primers at the bottom of the gel and streaking (Figure 2). When the annealing temperature was lowered, continued streaking was encountered, therefore the annealing temperature was raised to 52˚C for the sixth trial. The extension time was also changed to 45 seconds and different unknown DNA was used because the original IB-3 cells were depleted.
The sixth trial (Figure 3) yielded interesting results; the bands and streaking that was seen were too low on the gel to be considered usable as a definitive result; the location of these bands and the base pair length was determined by a logarithmic scale (Figure 4). These results were not able to be replicated because the unknown DNA was obtained from colleagues. Therefore the results could not be further investigated. The results of this trial did not support or refute our hypothesis. For the seventh trial new IB-3 purified genomic DNA from colleagues was used. The gel showed primers near the bottom, streaking, and nonspecific bands low in the gel for the wild-type primers. The results were inconclusive and the bands neither supported nor refuted the hypothesis (Wright et al, 2009). The eighth trial used IB-3 purified genomic DNA from colleagues. The annealing temperature was kept at 52˚C and the extension time was kept at 45 seconds. The gel showed no streaking and nonspecific bands in the mutant primer lane. All band obtained were not at 902 base pairs and are inconclusive and did not support or refute the original hypothesis.
The results did not support our predictions that primer 1, complimenting the G542X mutated nucleotides TGA, would anneal to the DNA of a person who carries the mutation. It does not support that DNA with the G542X mutation would not anneal to the control primer 2, which compliments the wild type nucleotides GGA. Primers for wild type and mutant did not express bands at 902 base pairs. These results did not confirm that primer 1 is a reliable assay for testing patients for the G542X mutation.
There are certain weaknesses in this test. The 1˚C disparity that exists between the annealing temperatures of the primers could lead to annealing failure and DNA amplification of the wrong sequence, or no amplification. The temperatures for annealing were adjusted during experimental trials, because optimal temperatures may differ from the values calculated. The mathematically predicted temperatures are only estimations of the ideal annealing temperature (Witter, 1993). This could be due to the varying annealing temperatures to account for the 1˚C difference between the primers. It is believed that the design of the primers caused the non-specific binding because the primers annealed to non-target areas of the DNA (Witter, 1993), which would explain the results of gels 6, 7 and 8. The sequences of the primers are correct, however if we had six more weeks, new primers could be designed that contained more base pairs. Therefore, the primers are more explicit to the certain area of the mutation G542X.
In our independent investigation, the history of the G542X mutation was explored as it traveled across continents. We researched the possible times when carriers of the mutation may have migrated from Mediterranean populations to Spain, and then again when a population of carriers brought the defect to the New World and Mexico, reproductively isolated them, and experienced a dramatic increase in the prevalence of the disease (Figure 5). We predict that this gene flow occurred during a period of heavy colonization by Spanish and Portuguese settlers, sometime in the 16th century during the Mexican conquest by Cortez (Clendinnen, 1991). We also investigated the current circumstances surrounding this disease in Mexican populations. We researched the current movement of the disease, by populations moving into America, legally or illegally, and what care options or agencies are available to sufferers both in areas of the United States where they might settle, and in the Mexican health care system (Figure 6). We interviewed Michigan State Professors about the health care system discrepancies between Mexico and the U.S. We researched recent immigration patterns from locations with high G542X mutation presence in Mexico and also the most accessible and most funded agencies currently treating CF in Mexico and in the Southern United States. We predict that the presence of the mutation in the United States will become increasingly noticeable due to the comparatively poor health care system and limited resources available in Mexico leading to more immigration to the United States, even though a discrepancy in treatment does exist for lower socioeconomic groups, like immigrants (Williams and Collins, 1995). Our findings show how a single mutation can affect different regions and populations (See Appendix for full analysis).
Beyond this experiment, there are other studies that could further contribute to the G542X mutation. Various ways to introduce wild type genes into mutated DNA, or gene therapy, is a prospective treatment for curing cystic fibrosis (Dettweiler and Simon, 2001) although there are a number of ethical and methodological objections to this field. Since the use of gene therapy in humans is still very much in the development and clinical trial stages, its use to treat diseases currently is very controversial as it serves as the only resort for many people, but the risk of the procedure is very high. The use of retroviral vectors to introduce the healthy gene to an organism can cause severe immune responses perhaps most notably, leukemia (Dettweiler and Simon, 2001). Additionally, using human subjects in clinical trials is quite dangerous since the mechanisms of gene therapy are still not completely understood and very little success has been seen in humans (Dettweiler and Simon, 2001). Knowing the location and primers that will locate the G542X mutation can help the experiments on gene therapy. Also, primers can be tested for heterozygous carriers of the G542X mutation, to be able to track the mutation before it has an effect on future generations of the carrier. Researchers of G542X have found no articles have been found relating to tests of the specific mutation itself, and said that future studies should devise an experimental procedure that could help educate people about the disease earlier in life to obtain the best treatment possible (Loirat et al, 1998).
Well 1 contains the 1 KB Invitrogen DNA ladder. Well 3 contains the mutant primers and well 5 contains the wild type primers. This gel shows that annealing of the primers definitely did occur, but since the bands appeared much lower in the gel than 902 base pairs, which they were designed to do, the results are likely incorrect. Well 5 contains a PCR reaction with the wild-type primers, and just as in well 3, significant annealing did occur, but not at the desired range. The annealing was likely to a similar DNA sequence at a different location on the DNA strand, and since the wild type and mutant primers are only one base pair different, they most likely both annealed to the same undesired sequence. The bands that appeared were determined to be incorrect by comparison to the known ladder DNA band lengths. They are visibly much too low, but in order to better identify the bands, a graphical representation was made (see Figure 4), and the lengths can better be estimated. Using the graph we were able to estimate that the lengths of the prominent bands in lane 3 range from about 450 base pars to 220 base pairs. Similarly the bands in lane 5 we predict to be from 470 base pairs to 220 base pairs. None of which are near the 902 base pairs bands that we were expecting (slightly above the 1018 mark labeled with the green arrow). We were never able to further analyze this DNA from the sample because it was taken from colleagues and only enough was received for one trial. If given more of this DNA, we could have ran a restriction enzyme test on it to perhaps better understand the undesired bands that appeared, or at least try and replicate these results. Similar results were seen for gels 7 and 8 that used different DNA, where bands appeared, but again lower than they would be expected, so no definitive conclusions could be drawn about their identity.