Inconclusive Detection of CFTR Mutation W1282X in Infected Human IB3 Cells Using PCR and Gel Electrophoresis

Authors: David Hart, Andrea Kulhawik, Brian Kim, and Megan Niner

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) mutation W1282X is a class 1 CF mutation, meaning mRNA is never produced or quickly degrades, resulting in the disease (Wilschanski et al., 1995). The mutation is a single base pair substitution from guanine to adenine at base 3978 on exon 20, making the original amino acid, tryptophan, switch to a stop codon (Shoshani et al., 1992). Polymerase chain reaction was used to create an indicative test to detect the presence of the CF mutation W1282X. Under standard PCR conditions with the designed primers and temperatures, Primers anneal to DNA from human IB3 cells and amplifies the mutation. The synthesized DNA was viewed using agarose gel electrophoresis. This showed a band with a length of 1,100 base pairs when using mutant primers because the primers were designed to anneal to the DNA with that distance separating them (Haenisch et al., 2009). Future CF researchers can view our experiment and results for comparison or reference. Different kinds of treatments for CF exist and gene therapy shows the most potential (Welsh and Smith, 1995). In order to test public opinions about gene therapy, several focus group surveys were conducted. For each trial, two groups were given a survey. One group was presented the survey, prefaced by a presentation providing background on CF and gene therapy. Another survey was given to subjects without additional knowledge. Through doing this, the effects of a population being more educated were analyzed using a X²-test. Overall results showed no significant difference between groups.

Figure

Figure 1: Amplification of IB3 DNA with an annealing temperature of 47°C. Three different PCR cocktails were made and were run through the thermal cycler at varying temperatures for 35 cycles. Initial denaturation occurred at 94°C for 1 minute, denaturation was run at 94°C for 45 seconds, primer annealing occurred at 47°C for 45 seconds, extension was run at 72°C for 45 seconds, and final extension was run at 72°C for 7 minutes. Lane 1 shows a band at 1100 bp for the mutant primer with some smearing. Lane 2 contains the wild-type primer and produced nonspecific binding. Lane 3 was the negative control with no template DNA, and lane 4 is the 1-KB ladder.

Discussion

Experiment Summary

Cystic fibrosis is a degenerative autosomal recessive disease caused by a mutation located on the long arm of chromosome 7 within the cystic fibrosis transmembrane conductance regulator (CFTR) gene (Frulloni et al., 2002). The most common mutation of CFTR is the DF508 mutation, which accounts for 70% of all cases (Welsh and Smith, 1995). Different mutations can cause a variety of symptoms including impairment of the intestines and liver, impairment and possible destruction of the lungs and pancreas, infertility, and mucus buildup, which causes bacterial infections (Welsh and Smith, 1995). The W1282X mutation is a specific variation on the CFTR gene at base pair 3978 in which a guanine is substituted for an adenine resulting in the original amino acid, tryptophan, being coded for a stop codon at amino acid 1282 on exon 20. This mutation is known to exist frequently in the Ashkenazi Jewish population, where W1282X accounts for 60% of cases (Shoshani et al., 1992; Haenisch et al., 2009).

Using PCR, mutations can be recognized and diagnosed. Because of the large amount of mutations, being able to differentiate is important for specialized and more effective treatment. There are many approaches for treatment, including gene therapy, drugs, and antibiotics (Welsh and Smith, 1995). When these treatments are fully developed, knowing which mutation is present will be important. Therefore, the purpose of our study was to successfully reproduce and identify the W1282X mutation to learn the mechanism as well as its importance in diagnosing and treating CF. In this experiment, a PCR reaction amplifying the W1282X mutation was designed and carried out with DNA from IB3 cells, which contain both the W1282X and DF508 mutations on separate chromosomes. It was expected that by using primers designed specifically to bind to the W1282X mutation under ideal PCR conditions that an indicative test for the mutation would be created. Because of this, we hypothesized that under standard PCR conditions with designed primers and optimal temperatures, the primers will anneal to DNA from human IB3 cells and amplify the W1282X mutation as well as the wild-type sequence.

In order to add a subjective aspect to a rather objective experiment, the idea of genetic experimentation and manipulation was explored. MSU students were surveyed about their opinions on different aspects of genetic screening and therapies. One group was provided with background information about cystic fibrosis while another group was not. By providing information to a group, it was believed that students may want to involve themselves more and support genetic experimentation as a method of treatment for cystic fibrosis. In modern medicine, the ability to run diagnostic testing in vitro for genetic diseases is a reality (Handyside et al., 1992). This reality will become more important as treatments are developed. Future generations will be affected by this process, causing opinions on these topics to change.

Original Predictions

Based on previous experimentation done with PCR, successful annealing of the specifically designed primers under ideal PCR conditions will result in a band visible through gel electrophoresis (Ling et al., 1991). The distance between primers 1 (mutant) and 3 (reverse) is 1,100 base pairs (bp), so that will be the length of the band. Primer 3 was also designed to work with primer 2 (wild-type) and produce a 1,100 bp band if the result is negative for the mutation, but positive for a wild type DNA sequence. Because IB3 cells contain the W1282X mutation on one chromosome and the DF508 mutation on the other, we predicted that there would be a 1,100 bp band for the mutant primer as well as the wild-type primer because it was designed to anneal to the same location as W1282X. For the survey, we predicted that the group that was given background information regarding cystic fibrosis would provide answers that would suggest that they would be more willing to support gene therapy as a treatment. This is because additional information and understanding of the topic may persuade the subjects of the survey to be more open minded. We also predicted that there would be dissimilar answers between the two different groups because the group that received information may answer more favorably towards treatments for CF, producing higher scores (from the 1-5 scale where the fifth choice was the most open or favorable to the specific question). Between science and music students, we predicted the science studies would be more open to genetic testing.

Results

Eleven PCR trials yielded inconclusive but increasingly consistent results. The first run, with an annealing temperature of 47° C, produced a clear band around 1,100 bp with minimal smearing in the well containing the mutant primer. The wild-type primer produced quite a bit of nonspecific binding (smearing), and the control was blank (Figure 1). The smearing may have been due to a too-low temperature, so the next step was to increase the annealing temperature to refine the both bands. Trials at 48°C, 49°C, and 50°C produced no mutant bands as well as nonspecific binding in the wild-type lanes. As the results continued to show faint, smeared bands, an extra micro liter (L) of magnesium was added in an effort to clarify them and run at 47°C. Another reaction with an extra buffer was also run at the same temperature for comparison. The reaction with extra buffer showed bands at approximately 1,100 bp and 500 bp for the mutant primer. Nonspecific binding appeared in both the wild-type primer and control lanes (Figure 4). For the added magnesium, faint smearing occurred for the mutant primer and control, and nothing resulted in the wild-type primer’s lane. Salt is a contributing factor to help DNA and primers bind together, but too much can inhibit binding. It is possible too much salt was present in the reaction since nothing but faint nonspecific binding was visible. Any results in the control are undesirable and may compromise results, suggesting contamination in the dye, PCR reaction tube, or gel. Otherwise, only the band at 1,100 bp supports the hypothesis.

Since extra magnesium or buffer did not significantly brighten or clarify bands, experimentation continued with the standard PCR reaction. Trial 10 was carried out with an annealing temperature of 46° C, and produced nothing in the control lane, bands for the mutant primer with the clearest at 1,100 bp, and non-specific binding for the wild-type primer (Figure 2). The final trial had an annealing temperature of 45° C, and produced the same results as trial 10 except for smearing once again in the control lane (Figure 3). The consistent smearing for the wild-type primer suggests nonspecific binding, which occurs when the annealing temperature is too low. However, its predicted temperature was slightly higher than the mutant forward primer due to the single base-pair change, and still produced smearing when the temperature was raised. A possible explanation for the lack of results is the significant annealing temperature difference between the wild-type forward and reverse primer. With more time and materials, a new reverse primer could have been designed with more guanine and cyanine content, raising the annealing temperature to be closer to the wild-type primer. The three bands showing consistently in the mutant primer lanes were possibly due to amplifying short segments of DNA similar to parts of the primer. The band at 1,100 bp suggests the primer is functioning and amplifying the W1282X mutation, supporting the hypothesis, but other factors including contamination in the control, the other distinct bands for the mutant primer, and nonspecific binding for the wild-type primer refute it. Finally, a sample of the template DNA was analyzed by a spectrophotometer. It showed the concentration of DNA was 57.129 ng/L. This confirmed the DNA was present and pure.

Focus Group Study

Based on the results of the first survey, it was revealed that question 5 suggested that the group provided with information would be willing to support therapy as a treatment. Also, this very same question showed slight differences between the answers of the two groups. Overall we concluded that providing information regarding cystic fibrosis to the public may not provide much support in the cause for gene therapy to be considered an effective treatment as the average of the answers were close to the neutral value. The survey was conducted once again with two groups of MSU music students, and each question also had a p-value determined from a chi-squared test (Table 2). Once again, only one question showed a significant difference to support the hypothesis. Question 1 shows a clear difference with the combined groups, with the group provided information having a higher score (P=.004) (Figure 6). Despite the overall neutrality in answers, comparing the results of Lyman Briggs students and music students proved to show a notable difference. For questions 4 and 5, the calculus students had considerably higher answers, supporting the prediction that the group with a science background would be more prone to support genetic treatments (Figure 5). Possible error with this survey includes that the results may not be the representation of opinions of all people. In order to compensate for these errors in the future, having an increase in the number of people who took the survey was necessary.

Future Direction

Given more time and materials, PCR trials would continue until definitive bands were produced. Since no wild-type band was produced after multiple PCR experiments, experimentation would continue trying to get a result for that. If raising the annealing temperature and cycles continues to yield no results, ordering new primers with closer annealing temperatures would be the next step. As another control to strengthen our supportive evidence, testing for the ∆F508 mutation would also be carried out. This would include designing primers, predicting temperatures and PCR conditions, and running trials as needed. In order to improve the results of our survey, we would intend to increase the sample size of each group. This will provide a more accurate conclusion from the survey and it would be comparable to society. Also, a factor in the similarities of the answers in the two groups may be because of the way the information was provided. Thus, if possible, another approach to presenting the information about cystic fibrosis may be tested in order to see if our prediction may still be plausible.