Genotypic Identification of the W1282X CFTR Mutation in Cystic Fibrosis

Using Allele Specific PCR with Human Buccal Cells

Team Nana

Rei Doko, Bailey Greene, Ashley Hare, Ben Mancini

Abstract

The W1282X mutation found in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is caused by a single base pair substitution (Castellani et al., 2008). This substitution changes the 1282^nd amino acid to a stop codon, preventing the full CFTR amino acid chain from being formed (Hamosh et al., 1991). The W1282X mutation represents 60% of instances of CF chromosomes in Ashkenazi Jews in Israel (Shoshani et al., 1992). The purpose of this research is to develop a polymerase chain reaction (PCR) assay with an original set of primers to diagnose the W1282X CFTR mutation. We hypothesize a patient can be diagnosed with W1282X using PCR because our designed wild-type primers will anneal only to DNA lacking the mutation at an annealing temperature of 66°C due to the primer base pairs not complementing its mutant allele (Soloviov et al., 2010). Using agarose gel electrophoresis, it can be determined which designed primer was successful in binding to the target sequence creating an amplified DNA product of 1024 base pairs after performing PCR. Amplification of the 1-Rz gene in Lambda virus through PCR was utilized as a positive control to assess the reliability of[HA1] the equipment, reagents, and procedures used in the assay. Published primers from Soloviov’s research were tested as a positive control for CFTR amplification after the Rz gene is successfully produced. Our study was able to successfully amplify a 178 base pair long segment of the human wild-type CFTR gene using primers published in the paper by Soloviov et al.. We predict that our designed primers will amplify a 1024 base pair strand on the CFTR gene because they complement and will bind to their target DNA sequences that enclose 1024 base pairs of the gene (Soloviov et al., 2010). This PCR-based assay can be useful to doctors and researchers for a more efficient diagnosis of cystic fibrosis. Our study contributes to the field of cystic fibrosis diagnostic research by designing unique primers and discovering procedural factors that strive to make diagnostic results more reliable.

Discussion

Experiment Summary

Severe cystic fibrosis is a caused by the lack of functioning CFTR proteins, which results in an inhibition of the epithelial cells' ability to transport chloride ions across the apical membraneane, and is particularly characterized by chronic infection and buildup of mucus along the airway. (Knowles and Durie 2002). The W1282X mutation is a nonsense mutation which substitutes a single base pair of guanine to adenine, changing the 1282^nd amino acid from a tryptophan to a stop codon (Hamosh et al., 1991). The premature stop codon prevents the protein from being fully synthesized and leads to premature degradation (Rowe et al., 2005).

Our team designed a PCR assay to detect a W1282X mutation in the CFTR gene in a sample of DNA to be used as a diagnostic tool. We hypothesize the presence of a W1282X mutation in the CFTR gene can be detected using PCR and gel electrophoresis with two allele specific primers -- one for the wild-type and one for the mutation -- and a common forward primer. Our assay is significant as it may be useful to medical professionals and researchers for quick diagnosis of patients with the W1282X mutation, and provides a template for other development of assays for other mutations (Castellani 2008).

Original Predictions

As a positive control, we will amplify a segment of the 1-Rz gene in lambda virus that is 395 base pairs in length (Taylor et al.. 1983). We predict that when the amplified DNA is run through gel electrophoresis, a band will appear next to the 395 base pair strand for the 1-kb plus DNA ladder because the Rz1F and Rz1R primers result in a 395 base pairs long amplification of the gene (Taylor et al.. 1983). Genomic purification of human buccal cells will provide DNA without cystic fibrosis. This wildtype DNA will provide a template for the published and designed primers. The published primers will be same set of primers published by Soloviev et al. We predict that the amplified wildtype DNA will produce a band of 178 base pairs and the mutant DNA will not produce a band when analyzed by gel electrophoresis because the primers that were published by Soloviev et al., were designed to bind to DNA without the W1282X mutation (Soloviev et al.,2010). The designed primers are designed to bind to both the mutant and wildtype DNA. We predict the wildtype DNA will produce a band of 1024 base pairs when amplified with the wildtype allele specific primer and no band when amplified with the mutant allele specific primer because the mutant allele specific primer is designed to only bind to DNA with the W1282X mutation (Hamosh et al., 1999). Seeing a band with the wildtype allele specific primer and no band with the mutant allele specific primer will show that the DNA amplified does not have the W1282X mutation. We predict the mutant DNA will produce a band of 1024 base pairs when amplified with the mutant allele specific primer no band when amplified with the wildtype allele specific primer because the wildtype allele specific primer is designed to only bind to DNA without the W1282X mutation. Seeing a band with the mutant allele specific primer and no band with the wildtype allele specific primer will show that the DNA amplified does have the W1282X mutation on exon 20 of the CFTR gene (Hamosh et al.,1999).

Ultimate Findings

PCR amplifying the Rz gene within lambda virus using Rz1F and Rz1R primers has been successful in past research experiments (Taylor et al.,1983). Our first trial was successful in amplifying the Rz gene through PCR (Figure 4). By using different concentrations of MgSO₄ we were able to determine that using 1.5µL of MgSO₄ is most effective for PCR, which is needed because magnesium acts as a cofactor, increasing the productivity of Taq polymerase (Lorenz 2012). Since this trial was successful, we are able to use it as our positive control because it verifies that the reagents used for the PCR reaction and gel electrophoresis were able to work properly. By knowing that these materials work, we are able to determine what is not working when PCR is unsuccessful by eliminating the materials as the problem (Lorenz 2012). Our first three PCR reactions using the published primers were unsuccessful (Figures 5 and 6). We found that the concentration of DNA used was not high enough and that the annealing temperatures were too low (Soloviev et al.,2010). After changing these two components of PCR, we successfully amplified 178 base pairs of the CFTR gene on exon 20 (Figures 7 and 8) (Shoshani et al.,1992). Since we were able to successfully amplify the purified wildtype DNA we are able to use these results as a positive control when performing PCR with our designed primers because we know the DNA can be successfully amplified (Lorenz 2012). In all of our trials there were bands that were lower than the 100 base pair mark on the 1 kb plus ladder (Figures 5, 6, 7, and 8). All of these bands were lower than 50 base pairs and were brighter than the streaking from the RNA contaminants in the gel when the purified DNA was run through gel electrophoresis (Figure 3). This indicates that the two bands below 100 base pairs are primer dimers (Desmarais et al.,2012).

Future Directions

After drawing conclusions from our diagnostic assay with the published primers, it was determined that streaking seen on gel analysis along with faint bands can be improved with further experimentation. Troubleshooting this issue appears to be feasible as an increased concentration of template DNA increased amplified band brightness according to our findings. The observed streaking below the 100 bp mark on the ladder was determined to indicate formation of primer dimers. They were calculated to be 18 bp to 60 bp long using our standardized equation. Primer dimerization also commonly occurs from an excess concentration of primers, a low annealing temperature, and a low volume of DNA template added to the reaction vessel (Desmarais et al.,2012). A follow up assay should include different treatments that test lower primer concentrations in the PCR cocktail, higher annealing temperatures, and higher concentrations of template DNA in the cocktail to find the optimal levels of each variable that produce the brightest bands with minimal primer dimerization. A future direction for the diagnostic assay would be to test the primers we have designed on a DNA template with the W1282X mutation. A problem with an assay that did not work completely was a twisted ladder. With limited time in lab (4 hours/week), gel electrophoresis had to be run at a higher voltage for a shorter time. When increasing the voltage midway through running the gel, the ladder became slanted and the molecular size was not readable

DNA primers were designed to complement the ends of the target DNA sequence before starting the amplification process. A total of 3 different primers were needed to run a diagnostic test for either a wild-type or mutant CFTR genomic sequence over nucleotide 3978 (Vidaud et al,. 1990). We designed the forward primers to anneal to the location of the point mutation with the single base pair mismatch. The forward primer that would anneal to the mutant is 5’-CCCTAAGTTATTGAAACGTTGTCACCCCC-3’, and the wild type specific varient of the primer will be 5’-CCCTAAGTTATTGAAACGTTGTCACCTCC-3’. The designed common reverse will be 5’-CTGCTCAACTGGTGCTTTTAATTTATTTGTC-3 and will amplify a 1024 base pair strand (NCBI 2017).

Figures

PCR amplification of the CFTR gene in the human genome from buccal cheek cells using published primers and varying DNA volumes and annealing temperatures. Twelve PCR were organized to have different volumes of DNA from buccal cells and a wide range of annealing temperatures. The PCR for each trial included an initial 5-minute denaturation stage followed by 30 cycles of the following: a denaturation stage lasting 30 seconds at 94°C, an annealing stage lasting 30 seconds at the designated temperature, an extension stage lasting 30 seconds at 72°C. After the 30 cycles the machine cooled down to 12°C before the samples were taken out. A 1.8% LB agarose gel was run at 200V for 25 minutes. Lane 1 contained 5µL of 1 kb plus ladder and 1µL of 6x concentration blue loading buffer, New England biolabs. Lanes 2-13 contained 5µL of PCR product and 1µL of the blue loading buffer. (B) The plot shows the successful amplification, as the bands in lanes 2, 4, and 5 were determined by178 bp, indicated by the green line. The migration distance was measured at 3.75 cm, and put into the equation, indicating that the band is at 178 bp. The orange lines represent the other bands beneath- at a molecular size of 50 bp and 18 bp. To avoid multiple bands, DNA concentration can be lowered as it may be due to RNA contamination. (C) The components of each well. Well 1 was the 1 kb+ ladder, and wells 2 through 13 have varying DNA volumes and annealing temperatures specified.