PCR and Electrophoresis Assay Accurately Identifying the G551D Cystic Fibrosis Genetic Mutation

By: Scott Backman, Steven Cox, Andrew Failla, and I Rin

Abstract:

We identified the G551D Cystic Fibrosis mutation using a specifically designed PCR assay. Within the study, we used DNA extracted from DF508 S9 cells and G551D mutated plasmids provided by Dr. Silvia Bompadre, from the University of Missouri. The technique for genetic amplification, Polymerase Chain Reaction (PCR), was utilized to scan our sample for the presence of the mutation. Because the G551D strain of Cystic Fibrosis is the third most common worldwide, it is important that an accurate PCR analysis is available (Zegarra-Moran et al, 2002). After running PCR, gel electrophoresis was completed, allowing us to analyze the bands that resulted from PCR. We hypothesized that the specific designed mutant and reverse primers would anneal to the G551D plasmid, while the wild type and reverse primers would anneal to the wild type DNA (Wittwer et al, 1993). Based on the bands found in the gels, we concluded that an accurate PCR and electrophoresis assay was achieved. Theoretically, this assay could play a key role in diagnosing the G551D mutation in Cystic Fibrosis, which would allow physicians to provide information for treating the disease. Because PCR can reveal one’s genetic makeup, we created a survey about genetic testing. From the responses of the survey, we implemented statistical techniques in order to quantify the responses, revealing the different attitudes of science versus non-science majors toward genetic testing. We hypothesized science majors to favor genetic testing, and non-science majors to be less accepting, because past studies have shown there is statistical correlation (Hietala et al, 1995). We concluded from the results, students who major in science and who took more science classes favor more genetic testing.

Discussion:

Purpose of research and hypothesis

In the United States, the most common lethal genetic disease is Cystic Fibrosis (Campbell et al, 2008). CF is caused when the chloride transport channels are defective or absent in the plasma membranes or when there is interference with the ATP binding sites on the CFTR protein, preventing proper channel function (Li et al, 1998). The disease is caused by the inheritance of two recessive alleles for Cystic Fibrosis (Campbell et al, 2008). There are many different CF genetic mutations that can transpire within humans, such as the G551D mutation. This mutation is the third most common CF mutation, with a 3.1% worldwide frequency among affected patients (Zegarra-Moran et al, 2002). More specifically, CF affects the Celtic population with a frequency of about 8 % (Zegarra-Moran et al, 2002). The G551D mutation causes the chloride channel’s activity to be strongly reduced due to improper ATP binding within the cell’s CFTR membrane protein, which causes symptoms of Cystic Fibrosis (Zegarra-Moran et al, 2002). The reduction of the chloride channel function arises from the problems associated with ATP. The less ATP that binds to the protein to power the pump, the less efficiency the cell will have in pumping chloride through the membrane.

The reduction of channel activity causes pulmonary dysfunction and pancreatic insufficiency, along with other associated symptoms previously mentioned (Bompadre et al, 2007). Due to the severity and prevalence of this gene mutation, a PCR experiment was designed in order to detect and amplify target strands of DNA where the mutation is located. The DNA was extracted from S9 Cystic Fibrosis cells, and a plasmid containing the G551D mutation, which was provided by Dr. Silvia Bompardre of the University of Missouri. Gel electrophoresis was then utilized to identify the DNA and observe whether it was wild type or mutant. The hypothesis of this research states that the designed primers would accurately identify and distinguish between wild-type and mutant DNA through PCR amplification, which would allow a diagnosis of a patient with any Cystic Fibrosis Disease especially the G551D mutation. Our hypothesis is based on the findings of amplifying other missense mutation strands using PCR and electrophoresis, such as the N103K mutation (Friedman et al, 1991). The electrophoresis from the amplified DNA with the G551D mutation is expected to display a positive band using the G551D forward and reverse primers, while no band, indicating a negative result, is expected using the wild type and reverse primers. Conversely, the electrophoresis from the amplified wild type DNA is predicted to display a positive band with the wild type and reverse primers, while no band is expected from use of the G551D forward and reverse primers. Electrophoresis executed on the N103K mutation after a PCR used a protocol similar to ours, which accurately identified whether or not the amplified DNA containing the mutation (Friedman et al, 1991).

Primer Design

Using the CFTR genomic DNA sequence, all primers required for diagnosis of G551D mutation were designed. Forward mutant primers and wild-type forward primers were designed similarly but only switching the nucleotide from G to an A (Witter et al, 1993). Two reverse primers were designed, which tested at different base pair lengths. The first reverse primer was created on an intron 1200 base pairs down from the forward primer and was only used with the S9 cell DNA. The second reverse primer was created differently. Since the G551D mutation was inserted in a plasmid, this DNA only has exons. Therefore the second reverse was designed 1019 base pairs downstream from the forward primers.

Genomic purification

The Cystic Fibrosis Research Lab at Michigan State University provided the DNA from the S9 cells. Using the DNA purification protocol, the DNA was extracted successfully. DNA purification utilized buffers which act as detergents to disturb lipid-lipid interactions, resulting in the destruction of the cell membrane (Tseng et al, 2001). The wash stage was applied in order to eliminate unwanted impurities and salts. After proteins, salts, enzymes and lipids are removed from the environment and DNA bonds to a column, elution was applied to isolate the DNA (Tseng et al, 2001). Then the DNA was run in electrophoresis and a band was present in the gel, confirming the successful extraction of S9 DNA (Figure 1). However DNA extraction from plasmids was quite different. The filter paper containing the G551D mutation plasmids was cut and sterile water was added. The DNA was also extracted successfully as a band was seen in the electrophoresis gel.

PCR Analysis

In figure 3, using the DNA of G551D mutation, PCR was run with different temperatures of 46°C and 55°C. The purpose was to know which temperature would anneal the primers to the plasmids more efficiently. As a result, the primers designed both annealed at the two temperatures. In lane 3 and 4 at 46°C, bands around 1019 were observed, suggesting that at a lower temperature nonspecific binding occurred and provided inaccurate results (Wittwer et al, 1993). However as the temperature was raised to 55°C, the lane with wild-type forward primer showed significantly decreased band appearance, whereas the lane with mutant forward primer still showed a clear and promising band. In order to investigate the temperature difference, another experiment with 56°C and 58°C was conducted (figure 4). In lane 1 the mutant primers were employed, but inconclusive results were obtained. There was an absence of a band in lanes 2 and 3. This could be due to the annealing temperature being too high resulting in inaccurate plasmid binding (Wittwer et al, 1993). In this study with G551D mutation, we concluded that the best annealing temperature is between 55°C and 56°C. However further investigation is needed to make this conclusion. Figure 3 is the control experiment in which the primers designed for detection of the G551D mutation are used with wild-type DNA. It was expected that the mutant primers for G551D would not anneal to the wild-type DNA. The chance of finding DF508 mutation in this DNA was high because most of Cystic Fibrosis mutations are ∆F508. Using wild-type DNA, PCR was run with both wild-type primers and the mutant forward primer with its reverse primer. In figure 3, two different annealing temperatures of 46°C and 55°C were employed. Nothing was observed in lane 1 using the mutant forward primer and its reverse primer, because there is a mismatch at the 3’ end of mutant forward primer. This is because G551D is the CFTR missense mutation at exon 11 in which a single nucleotide “G” is changed to “A” resulting in substitution of an amino acid from glycine to aspartic acid at codon 551 (Cutting et al, 1990). However in lane 2, using wild-type primers, a band appeared around 1200-bp indicating the primer designed for wild-type DNA was successful. As the temperature was raised to 55°C, no bands were shown suggesting the annealing temperature was too high. This was repeated again in Figure 4 with 48°C and 50°C. In lane 1, using the wild-type primers, a clear band was obtained, suggesting the correct primers anneal at this temperature as well. In lane 3 and 4, a temperature of 50°C was used. No band was observed, suggesting the best annealing temperature for S9 cell is between 46°C and 48°C. Using this PCR assay and procedure, it would allow people to accurately determine if they possess the G551D mutation resulting in Cystic Fibrosis.

Survey analysis

Given the potential of ethical issues arising from genetic testing, it is extremely important to find out the opinion of the public. Polling science and non-science majors allows for the results to stem from more than one perspective. We predicted the majority of students that are science majors would be in support of genetic testing. This prediction was based on the assumption that science majors will be more educated in the area of genetics, and non-science majors may not understand the mechanisms and consequences of genetic mutations. Similar surveys on genetic testing have been completed by Eleanor Singer stating those who are more aware of science news were more approving of the genetic testing (Singer, 1991). Religion can also influence opinions on genetic testing. According to Schwartz et al, women with higher spiritual ideals are less likely to be genetically tested for ailments such as breast cancer (Schwartz et al, 2000). In our sociological study of attitudes toward genetic testing, we investigated different backgrounds such as major, gender, age, number of science classes completed, frequency of religious practice. Figure 5, shows the results of each category with their attitude toward genetic screening. All the tests have a T value greater than 1.9, which suggests the data is statistically significant. Students who are science majors, students who take more science classes, and students who attend less religious service favored genetic testing more than the other groups involved, thus, supporting our hypothesis about genetic testing.

Future Studies

When performing PCR, error could occur from every aspect of the experiment such as usage of wrong primers in the reaction cocktail, not adding enough or adding too much reagents, contaminations in the PCR tube, and not being able to test with a wide range of temperatures. All of these could cause an abomination of the success of the PCR. In order to further confirm our results, a restriction enzyme test could be utilized. When completing the survey (see Appendix), people may not have taken it seriously, which would skew the results. Untruthfulness is also a possibility, which could also account for inaccurate data. There were also outliers in the data that makes the statistical analysis difficult. Outliers included a 23 year old and a 16 year old completing the survey. Also very few people who completed the survey attended religious services often, which could make the results inaccurate. Including a more diversified population in future surveys may result in a better understanding a public opinion beyond the collegiate level. For instance, extending the sample population to either regional or state. Further research on this topic is possible based on our study. A multiple mutation PCR assay could be synthesized, simplifying the process of detecting specific mutations within the Cystic Fibrosis afflicted. Identifying specific mutations may allow for easier treatment assigned by medical professionals, and provide more essential data for determining the frequency of the G551D missense mutation.

Figure 3: This gel demonstrates the presence of mutant plasmid bands after running PCR. All PCR cocktails run on this gel contained the mutant plasmids extracted from filter paper. Lanes 1 and 3 contained mutant plasmids and mutant primers. Lanes 2 and 4 contained mutant plasmids and wild-type primers. Lanes 1 and 2 were assigned an annealing temperature of 55˚C. Lanes 3 and 4 were assigned an annealing temperature of 46˚C. Bands appear in the wild-type band because the annealing temperatures assigned were too low. Due to a single base pair mismatch associated with G551D, there would be non-specific binding occurring at lower temperatures, allowing the wild-type primers to anneal to the mutant plasmids.