Castle Crashers

Abstract

The disease cystic fibrosis can be caused by several mutations, all of which create a faulty cystic fibrosis transmembrane conductance regulator (CFTR) (Kulczycki et al., 2003).  CFTR is a protein that acts as a chloride channel in the cell; cystic fibrosis is a result of decreased chloride ion flow. The particular mutation dealt with in this report is 1078delT; a deletion of thymine which occurs on exon seven of the CFTR gene. This deletion results in a frame shift, which disrupts amino acid sequence for the rest of the protein (Haenisch, et al., 2009).  Allele specific polymerase chain reaction, PCR, was used in order to test for the presence of the mutation in the cells of a cystic fibrosis patient. Two different primers were designed that will bind to either the wild-type (primer one) or mutated gene (primer two). The resulting PCR products were run through agarose gel electrophoresis in order to ascertain the presence or absence of the thymine mutation in question. We predict that our primers will create a more accurate test of the present of 1078delT mutation, and that the wild type primers will only bind to wild type DNA as well as the mutant primers will only bind to mutant DNA ( Campbell , 2005).  Error was reduced by the primer designed to bind to the wild-type, or normal gene. Other control tests were ran to test for a delta F508 mutation, the most common genetic mutation that causes cystic fibrosis. These were able to confirm more accurately the mutation and the presence of cystic fibrosis in patients. A DIY was conducted in order to investigate the sociological thoughts on cystic fibrosis.

Figure1: PCR Amplification of 1078delT mutation of IB3 cells with corrected Primers at 44 and 46°C.  Thermocycler settings consisted of a three-minute denaturation cycle at 95°C, followed by 35 cycles of 30s at 95°C, 30s at 44 or 46°C,  and 60s at 70°C.  Finally, each mixture was elevated 72°C for a five minute elongation period.  Lanes 2-5 all contain non-specific binding, but seem to have a band at ~200bp.  However, it is evident that the gel was not run long enough, as the ladder did not extend the full length of the gel.  Nonetheless, there is a brighter band at 44°C WT than at any of the others, and the band at 44°C Mut is absent, which raises some concern. 

 

 

Discussion

 

Summary and Hypothesis

Cystic fibrosis is an autosomal recessively inherited genetic disorder, most often caused by the deletion of three nucleotides at codon 508 in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel in the cell membrane (Welsh and Smith, 1995).  This deletion 3-bp omission causes a protein misfold, which causes the CFTR protein to be rejected by the cell and sent to the lysosome, resulting in no chloride channels in the cell’s membrane, disallowing the escape of chloride ions (Friedman et al., 1991, Kulczycki et al., 2003).  This condition, caused by the defective CFTR protein, has many life-threatening effects on organs and is known as cystic fibrosis (Welsh and Smith, 1995).  However, instead of the three base pair deletion, the object of focus in this study is 1078delT, a Thymine exclusion occurring on exon seven of the CFTR gene, which results in a frameshift mutation (Haenisch et al., 2009).  By using different primers to bind to different genotypes, whether mutant or wild type, our aim was to use polymerase chain reaction (PCR) to create a fast, safe, and accurate method to diagnose the 1078delT mutation.

Manual PCR and DNA Purification

The manual PCR and DNA purification done before the CF PCR experiment, along with the electrophoresis gels run of those procedures show an understanding of PCR, DNA purification, and agarose gel setup.  These three activities are vital to the correct identification of the 1078delT mutation, as a PCR control, which makes the actual process smoother and more accurate (Haenisch, et al., 2009).  Having a working knowledge of the processes needed means that the experimenters have a greater chance of positively identifying error or incorrect results (Orozco et al., 1993, Friedman et al., 1991). 

Primer Sequencing

            Originally, three primers were created in the lab to correctly bind to wild type and mutant tests for the 1078delT mutation. Three primers were also created for the wild type and mutant tests for the ΔF508 mutation as an experimental control since it is the most common CF mutation.  After 12 failed attempts at PCR with no bands present, it was discovered that the primers had been incorrectly made.  The forward primers had been created to prime in the wrong direction.  The forward and reverse primers need to bind to opposite strands of the targeted DNA and the polymerase proceeds between the two primers (Saiki et al., 1988).  New forward primers were created that primed in the right direction. 

CF PCR

            With the original, incorrect primers, we had calculated that the correct annealing temperature would be 46⁰C (see Methods section).  Our first attempt at PCR, using these incorrect primers, we miscalculated the amount of water needed in the cocktail, causing the cocktail to be too diluted.  Eleven more attempts were made with the incorrect primers.  As we did not yet know that the primers were incorrect, we assumed there was something wrong in the cocktail or with the annealing temperature.  For several of these attempts, we increased/decreased the annealing temperature within the range of 40-48⁰C (Figures %).  Above or below the proper annealing temperature, nonspecific binding can occur, sometimes resulting in no band at all (Rychlik et al., 1990).  Due to the importance of finding a very specific temperature, a large variety of temperatures were tested.  In some attempts, we increased the amount of Taq polymerase, from 0.2 µL varying up to 2 µL (Figures %).  For one attempt, we used our ΔF508 primers as a control to see if there was something wrong with our primers. Seeing as these primers were created to prime in the wrong direction also, the control did not do what was intended, which was to ensure that the experiment was correct, besides the 1078delT mutation primers.

            Our first attempt at PCR using the correct primers with IB3 DNA showed bands with indiscriminate binding (Figure 1).  With a more accurate annealing temperature or salt concentration and more time, indiscriminate binding would decrease and a clear band could be found to support a positive test for 1078delT (Ignatov et al., 2002).  As we raised the annealing temperature, the bands became more clear (Figures 2 & 3).

            Originally, we hypothesized that by using different primers to bind to different genotypes, whether mutant or wild type, we would use PCR to create a fast, safe, and accurate method to diagnose the 1078delT mutation. Using PCR, we planned on detecting the 1078delT mutation of cystic fibrosis.  It was believed that with the positive test to detect 1078delT, the primers would successfully anneal and result in a clear band in the electrophoresis gel. Our correct primers showed bands with indiscriminate binding, so we were mildly successful in our experiment.  The appearance of bands supports our hypothesis for a positive test to detect 1078delT.

Research Survey

            The research survey conducted dealt with the sociological aspects of genetic testing.  Using a χ²test for statistical significance with a significance level of .05, we found a p value .7238 between Lyman Briggs and James Madison students, and .1393 for the professors of the same Colleges.  Therefore, we can conclude that this is not significant, since the p value is too large, and we fail to reject the null hypothesis of no difference between schools (Bearden et al., 1982).  These differences can be seen partially due to the variations in mindsets and majors, resulting in differing opinions of genetic testing. 

Future Directions

            To continue our experiment, if given more time, we would run more PCR tests and agarose gels from cocktails with varying amounts of DNA and Taq polymerase.  We would also vary the annealing temperatures until the optimal temperature was found.  Then, if a clear band still had not appeared in the gel, a longer annealing time could be tested.  Once a distinct band had appeared using IB3 DNA, we could obtain DNA that contained the 1078delTmutated genotype and run PCR using the cocktail that resulted in a clear band.  This would test the effectiveness of our primers and PCR method.

            Our research survey could be extended by adding more questions about how people feel about getting genetically tested or genetically testing their children for CF.  The questions could then be surveyed on a larger scale, encompassing many more majors, such as English majors, law majors, etc, as opposed to just science vs. non-science majors.  We would also spread the survey to a much larger quantity of students.  This would better test whether there is a statistical correlation between major and views on genetic testing.

            In a biochemistry seminar information was learned about protein shapes and protein folding. The ‘R’ group in the amino acid is the determining factor in how a protein folds. From this we determined that we could figure out the alteration in amino acids that occur in our frame shift due to mutation 1078delT. From this new amino acid sequence we could determine the change in protein folding that occurs by our mutation. Studying this might give insight into how the disease alters the CFTR protein. More studies would have to be done to figure out the new structure for mutation 1078delT as well as other mutations.