An Assay Demonstrating a 591 bp Band for the wt CFTR Sequence at the A455E Locus in CF Patients using S9 Cells and PCR.

By: The Red Charmanders:

Amanda Brohman

James MacVicar

Hannah Nelson

Ashley Woodward

Abstract

An assay was designed to test the DNA of cystic fibrosis (CF) patients for the A455E mutation. We hypothesized that a single base pair mismatch caused by the presence of the A455E mutation at the 3' end of a designed wildtype primer, at the correct annealing temperature and salt concentration, will not anneal to the DNA (Strasberg et al., 1997). One set of primers (#1 and #2) was designed to anneal to the A455E mutation. Another set (#3 and #2) was designed to anneal to the wildtype CFTR sequence at the A455E locus with #2 serving as the reverse primer, and the last set (primers #4 and #5) was designed to anneal to the ΔF508 mutation serving as a control. After a polymerase chain reaction (PCR) was conducted, the amplified DNA was viewed using agarose gel electrophoresis. A 591bp band was identified, indicating wildtype DNA. This experiment demonstrated an alternative method for identifying the A455E mutation in CF patients. For a psychological analysis, Michigan State students were taught basic facts about CF while drawing different colored beads representing allele frequencies in CF patients worldwide and in French-Canadians. Students were surveyed before and after the drawings on their opinions of genetic tests. We hypothesized that those who are exposed more frequently to controversial ideas (like stem cell research or abortion) will have a better perceived understanding than those less exposed. A chi-square test was conducted to determine if major and opinion were independent of each other. The chi-squared value was 1.89 and the p-vale was between 0.5 and 0.8, thus our hypothesis was refuted and the null hypothesis accepted; supporting no correlation.

Discussion:

Summary

Cystic Fibrosis (CF) is the most common lethal, autosomal recessive disorder among Caucasians, with one in 2,500 live births having the disorder (Fulmer et al., 1995). CF is caused by mutations in the CTFR gene. These mutations cause mucus build up in the lungs and pancreas, pancreatic insufficiency, and respiratory infections (McKone et al., 2003). The most common CFTR mutation, DF508, accounts for 70% of the mutations worldwide (De Braekeleer et al., 1998). The A455E mutation, accounts for 0.1% of CF patients worldwide (Wallis, 2007). A455E makes up 3% of CFTR mutations found in Dutch populations and 8% in the French Canadian population of Saguenay Lac-Saint-Jean (De Braekeleer et al., 1997). At the DNA level, this mutation is caused by a cytosine changing to an adenine at nucleotide 1496 and an alanine to glutamic acid on the amino acid level at codon 455 (Tsui et al., 1993).

Five primers were designed and used two at a time, one forward and one reverse, in polymerase chain reactions (PCR), to correctly identify if a CF patient was homozygous or heterozygous for the A455E mutation or homozygous for the wildtype (WT). We hypothesized that a single base pair mismatch caused by the presence of the A455E mutation at the 3' end of a designed wildtype primer, at the correct annealing temperature and salt concentration, will not anneal to the DNA. In previous PCR studies, primers were designed in much the same way, focusing on the single-nucleotide polymorphism (SNP) found in A455E and amplified using PCR with the optimal annealing temperature and salt concentrations. This protocol resulted in correct amplification of the A455E mutation (Strasberg et al., 1997).

Knowing that the A455E mutation is most commonly found in the French Canadian population of Saguenay Lac-Saint-Jean (SLSJ), we were interested how the founder effect may have contributed to this difference in the populations (De Braekeleer et al., 1997). A probability experiment was created, in which the different allele frequencies of the worldwide CF population and the SLSJ population were represented with beads. We hypothesized that our experimental data would follow closely to the theoretical probabilities, based on the statistical nature of the chi-squared (X²) test (Urquhart et al., 2009). A comparison was also made between students’ majors and their opinion on genetic testing through a survey/interview. In a previous study it was determined that people who had heard or read more about genetic testing were more likely to view it as a socially beneficial procedure (Singer et al., 2008). Our hypothesis stated that individuals who are exposed more frequently to controversial ideas will possess different opinions than those who are less exposed. The psychological aspect of our study compared the students’ opinions of genetic testing before and after they were informed about CF. We hypothesized that students who possess more knowledge of controversial topics, like genetic testing, will be more inclined to accept these topics. To test these hypotheses chi-square tests were conducted on the survey data.

Results and Findings - PCR

To determine optimal conditions for this assay, multiple PCR tests were conducted with varying annealing temperatures (T_a), concentrations of S9 DNA, primers, and MgCl, MgSO₄, and Mg⁺⁺ mix(a mixture of MgCl and MgSO₄). We predicted that if the A455E mutation was present in the S9 cells, then using forward primer (FP) #1 and reverse primer (RP) #2 would result in a band of 591bp and if not present, FP #3 and RP #2 would anneal to the WT DNA and amplify a band of the same length. These predictions were based on previous studies that have replicated and identified CF mutations through primer design and the selection of correct T_a (Strasberg et al., 1997; Saiki et al., 1988). By using an optimal T_a of 38˚C and adding 1 μL of 5mM Mg⁺⁺ mix, a band at approximately 591bp was observed using WT FP #3 and RP #2 when the exposure on the camera was raised from 0.5s to 5s, indicating correct amplification but at a small amount. A 1Kb DNA ladder was used as a control to determine the length of the band (Haenisch et al., 2009). No band was observed when mutant FP #1 and RP #2 were used, supporting our hypothesis. It also supports our prediction that if the A455E mutation was not present in the S9 DNA, then the WT primers would anneal to the DNA and result in a band of 591bp. With this data, we can conclude that the designed primers were able to amplify a section of DNA at the desired length; however, we were not able to confirm that the segment was our desired segment. Further tests would need to be conducted to confirm this.

For a positive control using DF508, we originally predicted that with the use of FP#4 and RP#5 we would observe a band of 628bps. All that was observed for the ∆F508 mutation were band smears that were most likely the result of non-specific binding. With further testing on annealing temperatures and salt concentrations it is believed that the primers will be able to amplify the desired region and produce a band length of 628 base pairs.

Results and Findings – Statistical/Psychological analysis

For the statistical test, we predicted that we would draw a 0.1% representation for the A455E mutation in the worldwide population and an 8% representation for the SLSJ population (De Braekeleer et al., 1997). Using a X² test, the p-value for the representation of A455E in the SLSJ population was between 0.8 and 0.9. This value was larger than the critical value, 0.05, supporting the hypothesis. The test for the world wide population data did not yield the same results. Here the p-value was found to be <0.05, making it a significant test, rejecting the hypothesis. The expected values varied between different sources. The percent of DF508 mutation alleles varied from 70% to 75%, while the A455E mutation in the SLSJ population varied from 8% to 8.3% (Wallis, 2007; De Braekeleer et al., 1997; De Braekeleer et al., 1998). We chose to use 70% for DF508 and 8% for A455E as they were the most common values.

For the analysis of students’ majors and their opinions on genetic testing, we predicted that science majors would show more acceptance of genetic testing than non-science majors as the more frequently individuals are informed about genetic testing, the more likely they are to view genetic testing as favorable (Singer et al., 2008). A X² test for independence was run and a p-value of 0.05-0.1 ( >0.05) was calculated, signifying a failure to reject the null hypothesis that stated: major and acceptance of genetic testing are independent of each other. This refutes our original hypothesis. In a study testing opinions on reproductive genetic technologies (RGTs) - preimplantation genetic diagnosis (PGD), hypothetical genetic modification, and sperm sorting for sex selection - similar results were discovered. The researchers found that no matter the race, gender, or age of the participants, the participants agreed with RGTs when they would be implemented to avoid and/or detect deadly genetic diseases, such as CF (Kalfoglou et al., 2005).

For the psychological aspect we predicted that by educating participants about CF and genetic testing the individuals would show an increase in their approval of genetic testing. A X² test for independence was run and a p-value of 0.5-0.8 (>0.05) was calculated which results in a failure to reject the null hypothesis (no correlation), which rejects our original hypothesis. This could be a result of the short amount of time in between the questions. In another study, very similar to our own, conducted by Laskey et al., our prediction was refuted. This study was conducted on African American premedical students and their opinions on genetic testing. The students were given a survey before a summer course, which included lectures based on genetic principles and medical genetics, and then were surveyed after the course. The researchers found that there was little change in opinion after they attended the class (Laskey et al., 2003).

Further Direction

For the A455E mutation, a single band at approximately 591bp was observed using our designed primers; however, the band was very faint. This could mean that the concentration of the S9 DNA was too low and the PCR was unable to amplify enough of the desired sequence to be easily observable. In order to eliminate NSB, the T_a would have to be raised slowly with each additional test to locate the optimal temperature. When the T_a is too low in a PCR, primers are able to create hydrogen bonds with sections of DNA that only math part of the primer. This leads to the amplification of multiple sequences of DNA, creating multiple bands on the agarose gel like the bands we observed. If there was more time to conduct further research, multiple T_a would be tested until the optimal temperature was reached. Another way to determine if the band observed was truly from the desired sequence would be to implement restriction enzymes. Restriction endonucleases, originally found in bacteria, are enzymes that cut specific sections of DNA (Campbell and Reece., 2005). By using restriction enzymes that would blunt cut parts of our desired DNA sequence, the resulting gel would showcase the areas of the DNA that had been cut to different, known lengths. If the predicted band lengths were the same as the actual band lengths, it would show that the correct area of DNA had been amplified.

For the ∆F508 mutation, only smears appeared on the gels, most likely as a result of NSB with this mutation, which could be resolved in the same way as with the other mutation; by increasing the T_a. With more time we would look into different annealing temperatures to find the optimal. The use of restriction enzymes cannot be implemented until one solid band is observed for this mutation.

The NSB from both mutations could also be resolved if Yaku primers were created. Our assay used the Traditional primer design method which requires primers that are exact compliments to the template sequence. It is possible that the primers were designed incorrectly; however, they were confirmed by the National Center for Biotechnology Information’s Primer-Blast website. The Yaku-Bonczyk method of primer design adds an intentional base pair mismatch at the 3^rd or 4^th base from the 3’ end of a forward primer, making it harder for the primer to anneal incorrectly (Yaku et al., 2008). This could also act as a second control to prove that the band we observed was the amplification of the intended sequence.

Gel 9

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7

12,216 BP

2,036 BP

1,018 BP

506 BP

298 BP

Figure 2. Resulting gel after PCR tests on DF508 mutant DNA using primers 4 and 5. Lane 1 contained 5mL of 1 kb ladder and was a positive control. Lane 2 was our standard cocktail with no DNA added and was used as a negative control. Lane 3 had DF508 mutant DNA with primers 4 and 5, and there was nothing visible in the lane. Lane 4 contained 3mL of DF508 mutant DNA (1mL in addition to the standard cocktail) with primers 4 and 5, resulting in nonspecific binding. Lane 5 resulted in nonspecific binding and contained DF508 mutant DNA, primers 4 and 5, and mL of 5mM MgCl. Lane 6 contained DF508 mutant DNA, primers 4 and 5, and 1mL of 5mM MgSO₄, resulting in nonspecific binding. Lane 7 contained DF508 mutant DNA, primers 4 and 5, and 1mL of 5mM Mg⁺⁺ mix, and nothing was visible in the lane The annealing temperature for all of the PCR products was 38°C.