Detection of CFTR mutation W1282X in IB3 human DNA with a band length of 884 base pairs using PCR and Gel Electrophoresis.

By: the Pulmonary Pandas

Ashley Manning, Chelsy Mahon, and MinJae Kim

Abstract

Cystic Fibrosis, one of the most common genetic diseases affecting those of European descent, is a recessive disease that targets the wet epithelium of the body (Welsh and Smith, 1995). W1282X is one possible mutation among many that can result in the disease. This mutation is where the nucleotide Guanine is changed to Adenine, transforming the Tryptophan codon into a stop codon at amino acid position 1282 (Moran and Verkest, 2007). Primers were designed to assay successful replication of the sequence for the W1282X locus based on the Yaku method. Positioned on exon 20, a forward wild type primer and a mutant primer were designed to bind to the anti-sense strand, while the reverse primer was designed to be 884 base pairs away on the sense strand. Our hypothesis is that Yaku primers provide an additional base pair mismatch in comparison to the traditional single base pair mismatch, therefore the specificity of the reaction in the allele-specific amplification will aid in the detection of the targeted sequence for the mutation W1282X in polymerase chain reaction. Once the PCR process was completed, the replicated targeted DNA was then analyzed using gel electrophoresis to determine if the mutation is homozygous wild-type, heterozygous, or homozygous for the mutation. A band length of 884 base pairs was produced in separate lanes containing the PCR products; one with the forward wild-type primer and the other with the forward mutant primer. The sample was found to be heterozygous since it contained bands in both the wild-type and mutant lanes. In addition, we focused on the chemistry aspect of CFTR as we researched the ions transported via the CFTR protein. We hypothesized that ions with a similar size, and/ or charge to chloride will be transported through the channel of the CFTR protein. It was determined that the ions transported through the CFTR channel were all anions and as the radius difference increased from that of the chloride ion the permeation decreased.

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Figure 5 : Amplification of target DNA containing the mutation W1282X. The PCR conditions included pre-denaturation at 94°C for 5 minutes and 35 cycles of 94°C at 45 s, 36°C at 45s, 72°C at 45s and final extension at 72°C for 7 minutes. This figure shows the most successful PCR products taken on November 11^th, 2009. Lane one contains the ladder DNA, while lanes two and five contain wild-type primers (wild-type forward and reverse), lanes three and six contain mutant primers ( mutant forward and wild-type reverse), lastly lanes four and seven contain the control (removal of Taq polymerase). The successful post electrophoresis gel shows some non-specific binding in lanes three and six as lane as a band around 884 base pairs which is desirable for detecting the mutation W1282X. The bands in lanes two and five correspond with those in the mutant lanes, however this was not expected and may be because the DNA tested is heterozygous for CF mutations. Control was negative, showing no results.

Discussion

Experimental Summary:

For the purpose of this research we focused solely on the class I mutation W1282X , which occurs when the Tryptophan codon is transformed to a stop codon at position 1282. This results in the shortening of the CFTR protein specifically causing pulmonary disease and pancreatitis (Moran and Verkest, 2007). It was hypothesized that allele specific primers with the utilization of the Yaku method could be used to detect W1282X using Polymerase Chain Reaction for the amplification of DNA and gel electrophoresis for confirmation of W1282X via band length comparisons. In addition, the detection of the W1282X mutation research was also done on ion transportation via the CFTR protein. Specifically on what types of ions can be transported via CFTR and whether that was affected by charge, size or other periodic properties.

Original Predictions:

By amplifying the genome of IB3 human cultured cystic fibrosis cells, it was predicted that the DNA purification would yield 3 to 8ug of pure DNA based on this given amount for a million (the suggested amount) purified blood cells (Generation Capture Column Kit Manual). It is logical to assume that the DNA contained in a blood cell would be close to that of the cultured cells, and should therefore yield similar results. Ultimately, we expected to purify a large amount of DNA via the Generation Capture Column method. It was then predicted that the three created primers: wild-type forward, wild-type reverse, and mutant will help in successfully identifying our mutation W1282X. Each primer is 20 base pairs in length. Primers of longer length (20-30) are designed to reduce the possibility of the non-specific binding allowing for adequate specificity, and yet, are short enough to anneal to the DNA sequence (Haenisch et al., 2009). We ensured that the forward and reverse primer annealing temperatures did not vary by more than 2°C to create maximum efficiency in their binding to the CF DNA. If the temperature varied greatly between the primers, it would be hard to choose the correct annealing temperature, and therefore non-specific binding may occur. Based on the idea that primers placed further away are more effective, we placed the reverse primer 884 base pairs away from the forward wild type primer. If the primers were too close, the amplified product may be too small and run off the gel. (Haenisch et al., 2009). We further predicted that the mutation would be discovered at a band length around 884 base pairs compared to that of the ladder DNA injected into lane one . This is supported by the known lengths of the DNA ladder control and the placement of the reverse primer at 884 base pairs away from the forward. The prediction is based on the previous studies of Mike Moran and Nikki Verkest in which their reverse primer was placed 1,200 base pairs away and the detection of their mutation was found to be 1,200 base pairs in length compared to the ladder DNA control. We also supported these findings with our created DNA control. The negative control was in the PCR cocktail with the removal of Taq polymerase. We predicted that this lane would not produce bands because without Taq Polymerase no replication of the target DNA will occur (Haenisch et al., 2009). Lastly for our additional research we predicted that ions similar to chloride in size and charge, and even those in the same periodic group should be permitted through a normally functioning CFTR channel (Freeman, 2005).

Results and Findings:

The purification of the cystic fibrosis DNA is one of the most important steps in the detection of the mutation W1282X. If all contaminants are not removed the primers may not work due to the lack of available binding DNA, therefore rendering our bands inconclusive. The gel electrophoresis product of DNA purification yielded a bright band in correspondence to that of the most concentrated DNA in lane 4. This, therefore, confirmed our prediction that the DNA purification would yield a large amount of pure DNA (Figure 1). In order to determine optimal PCR conditions several experiments were preformed to vary cocktail (amounts) concentration, types of buffer, cycle times and most importantly annealing temperatures. Annealing temperatures directly relate to the effectiveness of the primers, and without effective primers the detection of the mutation W1282X would be unlikely. After several PCR products and gel analysis, the optimal annealing temperature was determined to be 36°C and the correct primers were created after six unsuccessful trials of PCR showing no results (Figure 2). It was also found that longer cycle times in pre-denaturation and extension were more effective for the amplification of DNA. The variation in cocktail amounts showed that 1μl instead of 2μl of primers and DNA was more effective in reducing primer dimmers and overly bright bands that made their analysis more difficult (Figure 3). The change in buffers from the given to the Yaku designed buffers showed no significant change in results of PCRs trials 1-12 with the normal buffers compared to the PCR products of 13 and 14 with the Yaku buffers (Figures 2-6). However, the bands were more distinct around 884 base pairs with the lack of a clear dependable ladder DNA (Figure 6). Therefore, the results of this gel are inconclusive and are not strong enough to use as evidence to support the hypothesis. Though it is possible the Yaku buffers did help in more specific binding, it is more probable that the optimal annealing temperature of 36°C was the source. Overall, the lack of accuracy experienced due to high instances of smearing, primer dimers, and one gel not having a clear ladder does not support our hypothesis.

The bands of expected length supported our hypothesis in that Yaku primers were used in the detection of the mutation W1282X. The appearance of the same band in both the mutant and wild-type primers suggests that the IB3 DNA may be heterozygous. Heterozygous means that the DNA examined would contain both the wild type sequence and the mutant sequence that the primers were designed to anneal to. Using the Cystic Fibrosis mutation database it was found that exon 20 contains other CF mutations close to the location of W1282X, therefore it is possible our wild-type seeking primers could anneal to another mutation as well as W1282X, accounting for the wild-type band. Similar support was found in the article by Friedman et al. that discusses the most common mutation ∆F508. The article discussed the heterozygous nature of ∆F508 due to the numerous mutations found on exon 10 and 11. Therefore, we can conclude that the mutation W1282X was successfully detected in the IB3 DNA using our designed primers.

Ion Transport and CFTR :

Research was provided by McCarty who found that ions within the same group as chloride, such as Bromine, Iodine, and Fluorine had some permeability in the wild-type CFTR protein. Bromine, Iodine and Fluorine all contain negative charges as does chloride, but vary in size (McCarty, 2000). Bromine has the same charge and a greater size than chloride but was found to be more permeable. Iodine is the largest of the elements tested, and was just below chloride in permeability. Lastly, Fluorine was the smallest element and had the least amount of permeability (McCarty, 2000). This supports our hypothesis in that the ions are the same charge as chloride, and those of the same periodic group would be conducted through the normally functioning CFTR channel.

Other work provides research that CFTR may also conduct bicarbonate ions that ultimately affect the pH of the epithelial luminal fluid (Poulsen et al, 1994). Poulsen et al. hypothesized that if CFTR conducted bicarbonate and the protein was mutated, then the pH of the luminal solution of the epithelial may be affected. Poulsen found that in an intact animal CFTR containing epithelial cells will secrete significant amounts of bicarbonate across the apical membrane. However because most epithelial cells have many methods of regulating bicarbonate it is unlikely that CFTR contributes a large amount to the regulation of pH. Poulsen et al. also suspected that bicarbonate is reduced in the mutant CFTR which may explain some of the pathology i.e. bacterial buildup and altered mucus that exclusively studying Cl cannot. In summary CFTR does conduct bicarbonate ions, whether that is directly mediated through the channel or through the movement of chlorine ions via exchange proteins we are still unsure, what is important to this research is that CFTR does conduct other ions that may contribute to the symptoms of the disease and other health related issues. Also this refutes our hypothesis that only ions similar in size, or in the same periodic group as Cl would be transported through the CFTR protein (charge is the same).

Lastly McCarty found that CFTR can conduct ions such as SCN, NO₃, acetate, glutamate, ClO₄, isethionate, and gluconate. Each of these ions have the same charge as Cl^-, but a radius larger than Cl^- (181nm< X). This supports the hypothesis that CFTR will conduct ions of similar charge, but not of size or of the same periodic group. Selectivity based upon ion size occurs through size effects on hydration energies, thus the size causes physical exclusion from the pore. Anion channels such as CFTR are selective for monovalent (one valence electron) anions over divalent (two valence electrons) anions( McCarty,2000). Finally, anion channels have mechanisms that make them selective for anions over cations. Ion selectivity essentially depends on the relative energy needed to dehydrate the ion and the energy gained through the interaction of the ion with the pore. Typically ion selectivity is measured by relative permeability and relative conductance. Relative permeability reflects the ease at which the channel can pull in the ion and therefore can be attributed to the dehydration energy. Relative conductance is a measurement of the affinity of the pore walls so that sticky ions exhibit a reduced conductance (McCarty,2000). A graph was constructed to compare the relationship between the permeation and conductance of the anions (Figure 7). As the permeation decreased the conductance varied, showing no correlation. McCarty organizes the permeability of those ions into three distinct groups. Ions such as Cl, NO₃ and Br have significant conductance while acetate, glutamate, and gluconate are too large to for easily through the pore, lastly SCN, I and ClO₄ are small enough to fit through the pore but inhibit the current (McCarty,2000). Since we are comparing ions to chloride in its role for the CFTR protein, another graph (Figure 8) was designed after calculating the radius difference between the ion (X) and chloride (.181). A R² value calculated for the correlation between the radius difference and the conductance (G_X/G_Cl) is .1586. For the correlation between the radius difference and the permeation (P_X/P_Cl) an additional R² value was calculated to be .7487. With a value closer to 1, the permeation decrease is correlated with the increase in radius difference. Overall, the hypothesis for the ion transport was refuted because ions and molecules larger than chloride can be transported through the normally functioning CFTR channel.

Future Direction:

To improve our experiment, the effectiveness of our primers and single base pair mismatch can be tested on DNA with the known W1282X mutation so that the effectiveness of the two methods can be further compared. Also, primers containing more G/C’s can be created to create a higher annealing temperature which can enhance binding efficiency (Haenisch, et al. 2009). To confirm and clarify the band length of 884 base pairs experiments using restriction enzymes, DNA sequencing and cloning can be done. Further research and experiments could be conducted in relationship to the importance of ions in other portions of CFTR analysis. Currently, our paper demonstrates the importance of ions in the CFTR protein but it may be useful, and interesting to explore how ions play into the PCR process as well as gel electrophoresis. If more means, knowledge and time was available actual tests with the CFTR protein using other ions of negative charge could be performed to further test and confirm the hypothesis. This research helps in further understanding in Cystic Fibrosis and will open up further questions to be explored about mutations that result in the genetic disease and transportation through the CFTR channels.