Diagnosing the 3849+10kb C>T Mutation of the CFTR Gene in Human IB3 cells using ASPCR and Electrophoresis

Team Bacon:

Gina Cherniawski

Daniel Smith

Alexander Ryktarsyk

Abstract

Cystic Fibrosis is caused by the mutation of the cystic fibrosis gene on chromosome 7, resulting in the mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein (Welsh and Smith, 1995). There are currently more than 1,600 known mutations of the gene, causing various mutations in the protein and a variety of symptoms as a result (Liang et al, 1998). The 3849+10kb C>T mutation of the CFTR gene is a point mutation. This change causes the addition of an 84 base pair exon from intron 19 into the functional protein sequence, reducing normal functioning CFTR (Highsmith et al, 1994).  The primary goal of this research was to design a Polymerase Chain Reaction (PCR) assay to detect the 3849+10kb C>T mutation of cystic fibrosis. DNA used in the PCR cocktail for control and designed experiments was extracted from human IB3 cells using a capture column.  As a control experiment, lambda DNA and E. coli experiments were run in order to determine if the PCR ingredients function properly and to troubleshoot future experiments. Additional control experiments were performed using published control primers from the Highsmith et al paper in order to replicate a previous assay that was successful in determining if tested DNA was mutated with 3849+10kb C>T. We hypothesized that by designing custom Yaku-Bonczyk primers and controlling annealing temperatures in PCR, it will be possible to detect the 3849+10kb C>T mutation of cystic fibrosis, based on previous research experiments (Highsmith et al, 1994). The amplified DNA is analyzed using agarose gel electrophoresis to determine if the mutation is present in tested patients. Our results using designed wild type primers with wild type DNA run at an annealing temperature of 50.4^oC show a band of about 550 base pairs. Results from mutant primers with wild type DNA at all annealing temperatures yielded no detectable amplification DNA. The success of this experiment would greatly aid in cystic fibrosis research by providing a unique assay in detecting the 3849+10kb C>T mutation. Due to this assay being more specific to the 3849+10kb C>T mutation, it can be used to give a more precise identification of this mutation in an infected person.

Figure 7: Amplified segment of DNA of human IB3 cells using designed healthy primers and varied annealing temperatures

This gel is the result of wild type DNA extracted from human IB3 cells run with custom designed wild type primers in a 1% LB gel run at 228 volts for 20 minutes. The custom wild type seeking primers were designed using the Yaku-Bonczyk method to only amplify 549 base pairs of the wild type DNA. Lanes 1 and 6 contain the 1 kb plus ladder and lanes 2 through 5 contain the PCR product run at varying annealing temperatures ranging from 50-56 degrees Celsius. Lanes 2, 3, and 4 show non-specific binding due to incorrect annealing temperatures. Lane 5 shows the clearest band, which is located just above the 500 base pair mark of the ladder.  The semi log plot shows the migration distance of the ladder and corresponds this distance to the proper base pair lengths they represent. A line of best fit is used to yield an equation that can determine DNA base pair length based on migration distance. In measuring the migration distance of the clear band in lane four, it can be determined that the base pair length of this band is 558. This indicates that PCR was run at the proper annealing temperature because the band appeared at the desired band length region.

Discussion

Experimental Summary

Cystic fibrosis is the most common disease affecting Caucasians in the world (Ratjen and Döring, 2003). It is an autosomal recessive disease that affects the lungs, pancreas, intestines, and liver (Welsh and Smith, 1995). The disease is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) (Welsh and Smith, 1995). When properly functioning, the CFTR gene permits CFTR protein channels in human epithelial cells to allow chloride ions to flow into and out of the cell. There are many different mutations that can cause this disease, and each mutation causes a different level of severity of cystic fibrosis by limiting the amount of chloride channels or by inhibiting the channels ability to transport the chloride ions (Welsh and Smith, 1995). Generally, Cystic Fibrosis will cause mucus buildup in the lungs and block ductal passages of the liver and pancreas. Severity of the disease and the symptoms experienced correlate to the different mutations of the CFTR protein (Chiba-Falek et al, 1998).

One particular mutation of Cystic fibrosis is the 3849+10kb C>T point mutation which alters the mRNA (Dugue´pe´roux and De Braekeleer, 2005). The 3849+10kb C>T point mutation causes an 84 base pair section of intron 19 to be included into the functional protein sequence (Highsmith et. al, 1994). This new exon contains a stop codon, which contributes to the decreased function of the CFTR protein at the apical membrane. The addition of this exon into the genomic sequence causes the chloride channel to become altered, ultimately hindering the amount of chloride that can travel in and out of the cell (Highsmith et. al, 1994). Patients who have this mutation tend to maintain normal salt levels, however the person may still show symptoms of cystic fibrosis (Chiba-Falek et al, 1998). It is due to the normality of the salt concentrations that makes this mutation difficult to diagnose.  Therefore, the only method that can be used to diagnose a person with the 3849+10kb C>T mutation of cystic fibrosis is through the analysis of their genomic DNA (Highsmith et al, 1994). We hypothesized that by designing custom Yaku-Bonczyk primers and controlling annealing temperatures in PCR, it will be possible to detect the 3849+10kb C>T mutation of cystic fibrosis, based on previous research experiments (Highsmith et al, 1994).

Original Predictions

Allele specific PCR was used to determine the presence of the 3849+10kb C>T mutation within a strand of DNA by analyzing the product with gel electrophoresis. Two forward primers and one reverse primer were designed using the Yaku-Bonczyk method in order to determine whether a patient has this mutation. The forward primers were designed to anneal to either mutant or wild type DNA. A reverse primer was also designed to anneal to DNA with either forward mutant and forward wild type primers. Both forward primers include a mismatch, three base pairs away from the 3’ end of the primer, as shown in figure 1. In addition, the final base pair at the 3’ end of the primer is specific to either wild type or mutant DNA, as also shown in figure 1 (Yaku et al, 2008). Wild type primers will only show bands when applied to wild type DNA. Mutant primers will only show bands when run with mutant DNA, as shown in figure 2. The expected result for both trials is the amplification of a 549 base pair strand because the same region of DNA is being amplified whether the mutation is present or not. Although this mutation includes an 84 base pair insertion, it does not change the length of the DNA strand. The 84 base pair insertion becomes relevant during splicing, where in afflicted patients, it is left in the gene sequence and affects the creation of the protein. A person who does not have this mutation will have a protein sequence in which the intron is properly and completely spliced out. The absence of a band when viewing the gel indicates that the primers did not anneal due to the extra base pair mismatch within the primer (Yaku et al, 2008). Positive tests with these trials will demonstrate that the primers are specific to either mutant or wild type DNA.

Final Results and Ultimate Findings

The success of this experiment is based on multiple successful trials through each stage. Each trial contributed to adjusted different aspects of the experiment, such as annealing temperatures, the number of PCR cycles run, and the amount of agarose in each gel. The annealing temperatures varied depending on the experiment being performed due to the difference in the melting points of the primers. In general, PCR only required 35 cycles to produce the necessary amount of product of target DNA. However, when using the designed primers, 45 cycles were necessary to obtain the preferred amount of amplified DNA segments.  It was determined through several trials that a 1% agarose concentration generates the most stable and clear gels.

The results from lambda PCR trials as shown in figure 5 indicate that the ideal annealing temperature was 51.5^oC for the 1Rz1F and 1Rz1R primers. This temperature produced amplified bands of approximately 494 base pairs which is consistent with the predicted results. The next successful experiment performed included control primers with wild type DNA extracted from human IB3 cells. Figure 6 demonstrates that an annealing temperature of 50.4^oC provides a band approximately 419 base pairs in length, which supports previous findings from the Highsmith paper. This result demonstrates that the sample DNA being tested does not contain the 3849+10kb C>T mutation.   

The final two experiments were conducted with both designed mutant and wild type primers on DNA from human IB3 cells. When running PCR using wild type primers, bands appeared at 558 base pairs using the annealing temperature of 50.4^oC as shown in figure 7. This base pair length is consistent with the expected result of 549 base pairs. These results support our hypothesis because they shows that the primers specifically amplified the target DNA they were intended to anneal.  This test required 40 PCR cycles in order to isolate enough product to be seen in the gel. The final trial run included designed mutant primers with wild type DNA. This PCR trial produced no bands in the lanes of the gel shown in figure 8. This data is consistent with the prediction that mutant primers will only bind to mutant DNA. Based on the data that only designed wild type primers will bind to wild type DNA, it was determined that the primers were specific to the intended DNA strands. This specificity is confirmed because of the single base pair mismatch within the forward primers. These allow each primer to anneal to only the designated strand.

Future Directions

Based on these findings, it is possible to further this research in order to provide more substantial results for the designed mutant primer. Due to the considerable cost of genomic DNA containing the 3849+10kb C>T mutation, it was not possible to obtain this DNA. Therefore, the mutant primers were only run with wild type DNA, and because they did not anneal to wild type DNA, it can be said that the designed mutant primer were specific to only mutant DNA. While this statement is true, it could be further supported through adequate research using 3849+10kb C>T mutant DNA, as this would provide one hundred percent confirmation that this assay was designed correctly. Once this assay is confirmed to work, the designed primers should be used in a blind study of DNA. Each set of primers would be used in PCR to determine if the DNA is mutated. The presence of bands at the 549 base pair location with mutant primers will indicate the presence of the 3849+10kb C>T mutation. The presence of bands at the same length when using wild-type primers suggests the absence of the 3849+10kb C>T mutation.

If additional time were available to continue research, it would be possible to redesign both mutant and wild type primers to include a higher G and C concentration. This in turn would cause a higher probability of the primers annealing correctly because the G to C bond is much stronger due it containing more hydrogen bonds. This would allow for a more reliable test for this mutation. Performing multiple trials of PCR would also aid in finding the optimal annealing temperatures of the designed primers. Although this method is not yet guaranteed to work, multiple successful trials would indicate the viability of the new primers to be used in future research and diagnosis of the 3849+10kb C>T mutation.