Successfully identifying the genotype of N1303K CFTR gene mutation using ASPCR on human buccal cells

By: Jery Camacho, Julia Riehl, and Jovonnie Storey

Abstract

The mutation N1303K accounts for 1.5% of cystic fibrosis (CF) diagnoses worldwide (Osborne et al, 1992). A base pair (bp) alteration of cytosine to guanine occurs at nucleotide position 4041, resulting in amino acid position 1303 to change from asparagine to lysine (Osborne et al, 1991). This change causes a defect in the processing and trafficking of the CFTR protein (Farhat et al, 2015), (Osborne et al, 1991). Allele-specific polymerase chain reaction (ASPCR) protocols were implemented to determine if the CFTR gene in patients was wild-type homozygous, mutant homozygous, or heterozygous. We hypothesized that single base-pair mismatch primers used in ASPCR will identify the genotype of the N1303K mutation based on the presence of amplified DNA displayed as bands after performing agarose gel electrophoresis (Friedman et al, 1991). Two positive control PCR assays, lambda Rz gene and N1303K published primers, were used to indicate if the PCR procedure for the designed primers would result in successful amplifications. The lambda Rz control resulted in a successful annealing of the Rz1F and Rz1R primers, which produced a band weighing 381 bp. The assays performed with published primers had not resulted in successful PCR amplification. Additional trials changing the amount of DNA template in the solution is believed to produce a positive gel electrophoresis analysis. Mutant and wild type primers were designed for identifying the N1303K mutation. The N1303K genotypes are to be determined by the primers designed and the band lengths they produce. We predict N1303K homozygous DNA will form bands at 250 bp using the mismatch primer, wild-type homozygous DNA will form bands at 550 bp using the non-mismatch primer, and heterozygous DNA will form bands at both 250 bp and 550 bp using both primers because the primers are each 23 bp long and the mismatch primers have 204 bp between them, whereas the wild-type primers contain 504 bp between them (Wu et al, 1989). With the test being inexpensive and effective, it is a reliable method for detecting the cystic fibrosis mutation N1303K and determining if the patient is a carrier. (Richards et al., 1993).

Discussion

Experiment Summary

Cystic fibrosis is an autosomal recessive CFTR gene mutation located on chromosome seven (Welsh and Smith, 1995). There are over a thousand mutations known to cause cystic fibrosis and the symptoms vary between patients (Welsh and Smith, 1995). These mutations cause an improper folding of the CFTR protein, resulting in the inability to function or destruction by lysosomes (Welsh and Smith, 1995). CFTR proteins are responsible for chloride transport in and out of epithelial cells, which regulate the salt to water balance within cells (Rowe et al, 2011). When CFTR proteins are absent, chloride ions cannot be transported. This impacts the amount of water that exits the cell, causing the mucosal lining of organs to thicken due to the lack of hydration (Rowe et al, 2011). Bacterial build up then occurs in the thick mucus, leading to persistent infections. The lungs, pancreas, and liver begin to degrade (Farrell et al, 2008).

N1303K is a cystic fibrosis missense mutation located in the second nucleotide binding fold of the CFTR gene (Osborne et al, 1992). This mutation results in a single nucleotide change of cytosine to guanine at position 4041 on exon 21. This alteration changes the amino acid asparagine to lysine at position 1303 (Osborne et al, 1991). This codon change causes the CFTR protein to misfold during its production, resulting in defective processing and trafficking of the protein which leads to degradation by lysosomes (Farhat et al, 2015). PCR is effective for diagnosing this type of genetic disorder, but the question being addressed with this research is if PCR can be utilized to detect the N1303K mutation in the CFTR gene from human buccal cells. We hypothesized that single base-pair mismatch primers used in allele specific PCR will identify the genotype of the N1303K mutation based on the presence of amplified DNA displayed as bands after performing agarose gel electrophoresis.

Original Predictions

ASPCR was to be used to amplify segments of the CFTR gene. These segments were then to be analyzed by agarose gel electrophoresis to determine if the N1303K cystic fibrosis mutation was present. Before N1303K mutations could be diagnosed, confirmation assays had to be run in the form of a positive control to ensure the PCR procedure being used was accurate and produced significant results. For our first control, we utilized the lambda Rz gene. We predicted the lambda Rz gene primers would produce a 395 base-pair amplification of the lambda Rz gene because the forward primer is 25 nucleotides long, the reverse primer is 23 nucleotides long, and there are 347 nucleotides between the two primers (Taylor et al, 1983).

For the second positive control, a set of forward and reverse primers obtained from Osborn et al. was utilized. We predicted these primers would bind to all of the DNA samples tested, mutant homozygous, wild-type homozygous, and heterozygous, because the mutant segment of the gene is located 265 nucleotides into the amplified 477 base-pair sequence and not in the primer (Osborne et al, 1992). Therefore, every gel performed using these primers would produce a band at the 477 base-pair location (Osborne et al, 1992).

Two distinct forward and reverse primers were designed to discern between the wild-type homozygous and mutant homozygous alleles through a single base-pair mismatch. The mismatch was positioned at the very 3’ end to reduce the possibility of the mismatched nucleotide being overlooked and the primer binding to a non-complementary sequence (Yaku et al, 2008). The mutant reverse primer is located 204 nucleotides away from the forward primer’s 3’ end (Zielenski et al, 1991). We predict the mutant homozygous CFTR genes will bind to the mutant primer since their nucleotides are perfect compliments to each other, allowing extension to occur (Wu et al, 1989). The wild-type forward primer was designed to contain the original cytosine nucleotide at the very 3’ end. Therefore, we predict the wild-type primer will bind to the wild-type homozygous CFTR genes due to the complementary nature of their 3’ nucleotides, allowing Taq polymerase to perform extension of the DNA sequence (Wu et al, 1989). The genotype would be defined as mutant homozygous if annealing of the mutant primer transpires. On the contrary, if annealing occurs with the wild-type primer, then the genotype of the DNA would be wild-type homozygous (Wu et al, 1989). We predict the N1303K heterozygous CFTR genes would produce bands with both mutant and wild-type primers because one of the chromosomes contains the specified N1303K mutation, whereas the other chromosome does not (Chavanas et al, 1996). Therefore, one chromosome would be complementary to the mutant primer and the other complementary to the wild-type primer. If both primers are being tested on the same DNA and gel electrophoresis produces a band at both designated base pair lengths for each allele, then that CFTR allele is heterozygous (Chavanas et al, 1996). Unfortunately, this portion of the experiment was not executed due to time constraints.

Lambda Rz Results

Three lambda Rz gene PCR trials were performed. The first trial was conducted manually, and the results did not indicate band illumination in rows 1 or 8 of the gel. Uncertainty with this trial could have been that our blue loading buffer was not inserted correctly. Another possible source of error is using too high of an annealing temperature, which may disintegrate the primers along with the repeated transfer of the PCR solutions between the water baths, which cools them off. The second trial was executed using an auto cycler instead of water baths. This trial did not produce PCR solution bands, but the 1Kb+ ladder was present. This failed trial can be attributed to the absence of MgSO₄, which increases the productivity of Taq polymerase. The third trial was again performed using an auto cycler. This trial displayed bands for the 1Kb+ ladder and the first PCR solution. It was confirmed that this was a successful lambda Rz band through the creation of a logarithmic plot. Using this plot, it was found that the band produced weighed 381 bp. Since, the lambda Rz gene’s total base-pair length is 395, an experimental error value of 3.66% was calculated.

Published Primers Results

Five trials using the published primers from Osborn et al were conducted. Each trial changed a single variable, while keeping the rest constant, to find a procedure that would result in a successful PCR. The first change involved using a temperature gradient for the annealing phase to determine which temperature binded the primers to the DNA template most effectively. The length of each phase was changed next to identify if the PCR process needed more time for denaturing, annealing, or extension. The last two changes involved the PCR solution: first removing the 10X TBE buffer, then removing the 50 mM MgSO₄. This was done to see if these ingredients were not necessary with human DNA and the “lab ready” primers. A lambda Rz PCR solution was included in the final trial to rule out the possibility of electrophoresis errors.

Future Directions

The first procedure that should be performed in future research is the successful amplification and identification of the published primers obtained from Osborn et al. It was discovered at the end of the research period that a greater quantity of 13.5 µg/mL human DNA template must be used in the PCR solution for bands to appear. When running gel electrophoresis to verify whole human DNA was extracted from buccal cells, a minimum of 20 µL had to be added to wells for a band to appear. Therefore, additional trials should be ran using 3 µL of 13.5 µg/mL human DNA template in the PCR solution. The thermocycler should be ran for 30 cycles with denaturing at 94℃ for 1 minute, annealing at a gradient of 50℃-58℃ for 45 seconds, and extension at 72℃ for 1 minute and 30 seconds. In addition, the total volume pipetted into the wells should be increased to 24 µL and gel electrophoresis should be ran at 195 volts.

Once the published primers have been successfully amplified, the designed primers need to be tested. The procedure from the published primers trial that yields a successful PCR amplification should be the initial ingredients for the designed primers. The thermocycler should run for 30 cycles at 94℃ for 1 minute and 30 seconds to denature and at 72℃ for 1 minute and 30 seconds to extend. The annealing phase should last 45 seconds at different temperature gradients for each set of designed primers: 58-66℃ for the wild type primer and 54℃-62℃ for the mutant primer. This assay should also begin with 3 µL of 13.5 µg/mL of human genomic DNA, followed by increments of 1µL. Furthermore, a total of 24 µL should be added to each well.

Figures

Figure 5: Amplification of wild-type DNA using published primers as a positive control by PCR and analysis using gel electrophoresis. To serve as a positive control, the published mutant primers were added to wild-type genomic DNA. The PCR cocktail was first made containing Taq polymerase, 10 mM dNTPs, wild-type genomic DNA template, 10X TBE, 25 nmol forward primer, 25 nmol reverse primer, 50 mM MgSO4, and nuclease-free H2O. After preparing the master mix, two 50 µL volumes were taken and put into two separate microfuge tubes. The microfuge tubes were then placed in a thermocycler for 24 cycles of denaturing at 95.5℃ for 45 seconds, annealing at a gradient of 48.4-50℃ for 30 seconds, and extending at 72℃ for 90 seconds. Since a gradient was used, the annealing temperatures for each trial were different: trial 1 at 50℃, trial 2 at 49.8℃, trial 3 at 49.6℃, trial 4 at 49.3℃, trial 5 at 48.8℃, and trial 6 at 48.4℃. After performing PCR, a 1.5% agarose gel was made using agarose powder, 20X LB (Lithium Borate) buffer, distilled water, and SYBR-safe dye. Each PCR solution was mixed with blue loading dye. These mixtures were loaded into wells 4 and 6. 1Kb+ ladder was also mixed with blue loading buffer and was loaded into well 8. The gel electrophoresis process was run for 25 minutes at 190 volts. The gel was then observed under UV light to determine if the PCR process was successful. Since no bands were produced for the PCR solutions containing the published primers, additional assays need to be performed with alterations to the PCR solution and cycling temperatures and times. The 1Kb+ ladder and lambda Rz gene serve as indicators that the well loading and gel electrophoresis were carried out successfully.