Amplification of ΔF508 Mutation in CFTR Gene in Homo sapiens Using Allele Specific PCR and Gel Electrophoresis

By: Sean Cannon, Gabi Meitler, Ben Salva, Abby Struble, Taylor Van Deer

 

Abstract

 

The cystic fibrosis ΔF508 mutation is found in about seventy percent of patients diagnosed with the disease (Welsh and Smith, 1995). On the long arm of chromosome 7, when CTT is deleted at the 508th codon, phenylalanine is lost, creating dysfunctional CFTR proteins Welsh and Smith, 1995). The purpose of the project was to match and mismatch wild-type and mutant primers with wild-type and mutant CFTR genome, to be able to identify the presence of healthy versus the ΔF508 mutation in samples. We hypothesized that our custom primers designed using the Yaku-Bonczyk method would anneal better to the DNA than our custom primers without Yaku-Bonczyk due to the intentional mismatch on the 3’ end of the primers. After identifying wild/mutant-type sequences, we determined that the wild/mutant primers would anneal to generate a band of 538 with wild type DNA and 537 with mutant DNA.  Genome purification was performed on our own saliva using the Chelex Extraction Method (Emerson, 2012). Subsequently, a PCR cocktail was made using two of four primers, nucleotides and taq polymerase in a thermocycler set at 95ºC denaturation, 48ºC annealing, and 72ºC extension to examine the band length of the replicated DNA by using gel electrophoresis, which was run at 200V.  We also developed a sociological experiment that analyzes frequency of performed actions typical of cystic fibrosis patients, like sanitizing their hands and using nebulizers. We predict the band length of the wild-type will be 538 nucleotides long and mutant will be 537, because our researched and designed primers were employed to detect a specific complementary section of their respective strands (Garibyan & Avashia, 2013). For control genomic purification of wild-type DNA, we measured a testable concentration of 0.35mg/mL.  After testing all the designed primers, all bands generated were incorrect length due to insufficient primer design.  Regarding our sociological experiment, the frequency of washing hands and sanitizing was far greater for the individuals living as though they had cystic fibrosis, and we found that psychologically using a nebulizer in public was a difficult task to perform.

 

Discussion

Summary of Experiment

Cystic Fibrosis is an autosomal recessive genetic disease affecting over 30,000 people in the United States, with over 70% of those cases being linked to the ΔF508 mutation (Bobadilla et al., 2002). Many researchers are especially interested in studying the ΔF508 mutation because it is the most prevalent, severe, and fast-acting mutation that causes Cystic Fibrosis. We used PCR and gel electrophoresis in this experiment to create a diagnostic assay for the ΔF508 mutation to allow detection in samples of saliva in Homo sapiens. We utilized the lambda virus Rz gene as a control for PCR and gel electrophoresis. The intentional mismatch on the 3’ end of the primers created using the Yaku-Bonczyk method led us to hypothesize that our designed Yaku mutant primers would anneal better to the target DNA than our normal mutant primers created without the Yaku-Bonczyk method (Wittwer, et al., 1993).

Our experiment also used sociological testing of healthy people and put them into situations typical of cystic fibrosis patients, through use of a nebulizer, collection of hand-washing data and surveys. We hypothesized that in the sociological experiment, the healthy subjects would feel socially isolated from their peers and raise self-awareness for germs and how difficult it is to constantly prevent themselves from spreading germs (Saimon & Siegel, 2004).

Original Predictions

We predicted that the use of four designed allele-specific primers would allow us to identify the ΔF508 mutation in the CFTR gene sequence of multiple human genomic samples of DNA using PCR, because the primers would anneal at the ends of the target sequence and replicate the desired DNA, showing bands of 538 and 537 base pairs on the gel with the wild-type and mutant primers, respectively (Garibyan & Avashia, 2013). The use of allele-specific primers and qualitative PCR identifies the presence of a pathogen or disease, such as ΔF508. The presence of the mutation was verified by analyzing the gel when a band of DNA appears that is the same size as the DNA target sequence (Garibyan & Avashia, 2013). We predicted that the wild-type forward primer would only bind to wild type DNA and the mutant primers would only bind to the mutant genotype. Both annealing at the 79,605th base pair on the CFTR genome, they would present a band of 538 (wild type primer) or 537 (mutant primers) base pairs in length, because the single base pair mismatch on the 3’ end of the primer creates a discriminating primer that increases the likelihood of annealing (Wittwer, et al., 1993). We predicted that the mutant primer created using the Yaku-Bonczyk method would anneal better to the target mutant DNA sequence than the mutant primer created without the Yaku-Bonczyk method due to the presence of an intentional mismatch on the 3’ end of the primer, as mentioned above (Wittwer, et al., 1993). We also predicted that the use of MgCl2 would allow a band to appear because it optimizes the PCR reaction (Kramer & Coen, 2001). Regarding the lambda virus Rz gene control experiment, we predicted that the provided forward and reverse primers would anneal to the target DNA sequence and created a band of 359 base pairs (Chen & Richardson, 2018).

In the sociological experiment, we predicted that the handwashing recorded by the subjects that sanitized similar to a cystic fibrosis patient would wash their hands more often than the average healthy student, because hand washing is one of the most important sanitation techniques to prevent the spread of germs and infection (Saiman & Siegel, 2004). Also, we predicted that the use of a nebulizer would create elicit a feeling of rejection and distress in peers because of their inability to hide their disease and the interruption the disease causes to their daily activities (Devins, 1994).

Results and Ultimate Findings

            After several runs of PCR, the optimal annealing temperature was determined to be 48.7°C in order for the primers to anneal properly. If the annealing temperature is too high, the yield of the PCR will be poor; if the temperature is too low, non-specific DNA fragments can be amplified (Rychlik, et al., 1990). The lambda virus Rz gene control was used to find which components are necessary for PCR and the creation of a band. After many trials, bands appeared for both the DNA ladder and Rz gene; however, the DNA ladder has very bright and thick bands, so the migration graph may not yield accurate lambda band lengths (Figure 2). Thus, we do not know if it elongated to the expected 395 base pairs (Chen & Richardson, 2018). From these control experiments, we determined that MgCl2 was necessary for Taq Polymerase to bind properly to the primer (Kramer & Coen, 2001). The literature wild type primers yielded a band of 1811 base pairs, not the expected length of 61 base pairs, possibly because the literature’s scientists did not use the Yaku-Bonczyk method (Bartoszewski, et al., 2010) (Figure 5). The primers could have annealed to different places on the human genome due to a lack of an intentional mismatch.

The designed wild-type primer and universal reverse primer made a band of about 90 base pairs with target wild-type DNA, not the expected 538 base pair long band (Figure 6a). Both the normal and Yaku mutant primers annealed to wild-type DNA even though a base pair deletion occurs in the mutant primers (Figure 6a). Oddly, the two mutant primers elongated with wild type DNA to about 100 base pairs each, which could signify primer dimers or self-polymerizing RNA. The measured absorbance at 280 nm using the spectrophotometer signifies the quantity of RNA, so a high absorbance count at 280 nm denotes more RNA than needed and those high amounts could cause the RNA to self-polymerize. Additionally, the normal mutant primer with mutant DNA did form a band of about 90 base pairs in length, which was not the expected length of 537 base pairs. The Yaku mutant primer did not form a band with mutant DNA possibly due to inefficient primer design (Figure 7). When these primers annealed to wild-type or mutant DNA and elongated to a different length than expected, the primers may have annealed to a different spot on the genome than intended or annealed to themselves, thus elongating to varying lengths. Regarding DNA purity, the 260/280 measurement signifies overall quality at 1.088 for wild type DNA, while 1.7 is ideal. Lacking DNA purity could have caused problems with annealing. Our hypothesis was not supported because the Yaku mutant primer did not anneal better to the mutant DNA. Ultimately, we cannot determine which primer design was the most effective because the base pair lengths are all different than the expected results.

Our experiment found that there are many sociological effects on patients with life-threatening diseases, such as cystic fibrosis. The data comparing the sanitization habits such as hand-washing and use of hand sanitizer between healthy and diseased people confirms the idea that sanitization is essential in prevention of the spread of disease (Saimon & Siegel, 2004). The data collected showed that the use of hand sanitizer was more prevalent in CF patient sanitization than hand washing (Figure 8). This was due to a difficulty accessing a bathroom or sink when sanitization was necessary. In addition this experiment found that the use of a nebulizer in a public area created a feeling of distress in the subjects. Previous research has discussed how disruption of daily activity and lifestyles by an illness can affect patients’ psychological well being (Devins, 1994). The subjects did not observe as many glances when using the nebulizer as expected, and they did not feel separated from the other nearby students. Ultimately, the subjects’ awareness of germs surely increased through this experiment, and the subjects did not feel socially isolated but rather uncomfortable when using the nebulizer in public places. Below is the link to our video summarizing our sociological experiment:

https://www.youtube.com/watch?v=BxX6KJXxRgM

 

 

 

 

Designed primers against Mutant DNA. (A) The designed primers: wild, normal mutant, and Yaku-Bonczyk mutant were run with the universal reverse against wild and mutant PCR in the same gel. Wells 1 and 8 contain the 100 bp ladder. Well 2 has the wild primer with wild DNA PCR sample. Well 3 has the wild primer and mutant DNA sample. Well 4 has the Yaku-Boncyzk mutant primer and wild DNA. Well 5 has the Yaku-Bonczyk mutant primer with mutant DNA. Well 6 has the normal mutant primer with wild DNA. Well 7 has the normal mutant primer with mutant DNA. Bands appeared in wells 2, 4, 6 and 7. The gel was run for 225 V for 10 minutes using the 1X LB buffer.  The PCR master mixes included 160 uL nuclease free H20; 4 uL forward primers (wild, normal mutant or Yaku mutant); 4 uL of forward primer and 4 uL of universal reverse primer; 20 uL PCR buffer; 4 uL Lambda DNA; 4 uL Taq Polymerase; 4 uL dNTPs; and 14.80 uL MgCl2. 2 10 uL samples of PCR cocktail ran through the thermocycler at the following conditions: 95 ℃ for an initial 3 minutes; 95℃ for 30 seconds; 48.8 ℃ for 30 seconds; 72℃ for 1 minute; completed a total of 35 cycles. 7.5 uL of PCR sample and 1.5 uL of dye were inserted into the wells, along with 7.5 uL of ladder and 1.5 uL of loading dye (ingredients: bromophenol blue and glycerol). (B) The migration graphs shows the the bands that appeared were all 90 bp in length. The bands that appeared may have occurred due to contamination or high 280 count, so there may have been a lot of self-polymerizing RNA.